Production of red blood cells and platelets from stem cells

ABSTRACT

This disclosure provides methods of making a megakaryocyte-erythroid progenitor cell (MEP), comprising differentiating a stem cell into a MEP in culture in the presence of an aryl hydrocarbon receptor (AhR) agonist. In some embodiments the stem cell is a pluripotent stem cell. In some embodiments the MEP co-expresses CD41 and CD235. In some embodiments the number of MEPs produced in the culture increases exponentially. Methods of making a red blood cell (RBC) by culturing a MEP in the presence of an AhR agonist are also provided. Methods of making a megakaryocyte and/or a platelet, comprising culturing a MEP in the presence of an AhR modulator are also provided. In some embodiments the AhR modulator is an AhR antagonist. This disclosure also provides compositions comprising at least 1 million MEPs per ml and compositions in which at least 50% of the cells are MEPs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/683,246, filed, Aug. 15, 2012, which is hereby incorporated herein byreference

GOVERNMENT FUNDING

This invention was made with Government Support under Contract Nos.HL107443, ES11624, and ES007381 awarded by the National Institutes ofHealth. The Government has certain rights in the invention.

INTRODUCTION

Blood transfusion is an indispensable cell therapy, and the safety andadequacy of the blood supply are national and international concerns. In2009 alone, the National Blood Data Resource Center reported thatblood-banking institutions collected more than 17 million units of wholeblood and red cells with hospitals in the US transfusing over 15 millionpatients yearly. Due to substantial polymorphisms of blood groupantigens, there are, even in developed countries, chronic shortages ofblood for some patient groups. In the US, more than 40% of Sickle CellAnemia patients, who are largely of African descent, experience immunereactions when transfused with blood from donors, who are mostly ofCaucasian decent. Sporadic shortages of blood can also occur inassociation with natural or man-made disasters. There is also increasingconcern that the blood supply may be curtailed by new restrictions ondonor eligibility as new blood transmissible diseases are discoveredand/or emerge and spread to new geographical locations. Lastly, bloodusage by the growing numbers of individuals greater than 60 years of ageis predicted to increase, leading to an insufficient blood supply by2050.

For these and other reasons, there is a need in the art for new methodsof making red blood cells and platelets. There is also a need for newmethods of making myeloid-erythroid progenitor cells (MEPs), which forexample enable production of red blood cells and/or platelets.

SUMMARY

This disclosure provides, among other things, new methods of culturingstem cells, including pluripotent stem cells, under defined, feeder freeconditions in the presence of AhR ligands to cause outgrowth of MEPs, apopulation of bipotential myeloid-erythroid progenitor cells (MEPs), aswell as methods of differentiating MEPs into red blood cells,megakaryocytes, and platelets. In some embodiments the methods allow forthe exponential expansion of MEPs in culture and production of almostunlimited quantities of red blood cells and platelets. According, thedisclosure also provides compositions of at least one of MEPs, red bloodcells, megakaryocytes, and platelets, and methods of using those cellsto treat a patient in need of transfusion with red blood cells orplatelets, such as sickle cell anemia patients. The disclosure furtherprovides methods of screening a compound for an effect on a red bloodcell, megakaryocyte, platelet, or MEP. Those methods find use, forexample, in identification of drug product candidates and screeningmolecules to identify potential toxicity. These and other aspects ofthis disclosure are provided herein.

In a first aspect this disclosure provides methods of making amegakaryocyte-erythroid progenitor cell (MEP). In some embodiments themethods comprise differentiating a pluripotent stem cell into a MEP inculture in the presence of an aryl hydrocarbon receptor (AhR) agonist.In some embodiments of the methods the culture does not comprise serum.In some embodiments of the methods the culture does not comprise feedercells.

In some embodiments the methods comprise differentiating a pluripotentstem cell into a MEP in culture in the presence of at least one proteinselected from BMP-4, vVEGF, WNT3a, bFGF, hSCF, FLT3, TPO, and EPO. Insome embodiments the methods comprise differentiating a pluripotent stemcell into a MEP in culture in the presence of BMP-4, vVEGF, WNT3a, bFGF,hSCF, FLT3, TPO, and EPO. In some embodiments the differentiationculture further comprises an aryl hydrocarbon receptor (AhR) agonist.

In some embodiments the methods comprise a) culturing the pluripotentstem cell in RPMI media supplemented with BMP-4, VEGF, Wnt3a, andknockout serum replacement (KOSR); b) culturing the cell obtained fromstep a) in RPMI media supplemented with BMP-4, VEGF, bFGF and KOSR; c)culturing the cell obtained from step b) in StemPro 34 mediasupplemented with BMP-4, VEGF, and bFGF; d) culturing the cell obtainedfrom step c) in StemPro 34 media supplemented with VEGF, and bFGF; e)culturing the cell obtained from step d) in a mixture of IMDM and HamsF12 supplemented with B27, N2-supplement, BSA, VEGF, bFGF, hSCF, andFlt3 ligand; and f) culturing the cell obtained from step e) in amixture of IMDM and Hams F12 supplemented with B27, N2-supplement, BSA,VEGF, bFGF, hSCF, Flt3 ligand, hTPO, IL-6, and EPO. In some embodimentsthe culture in step f) further comprises a AhR agonist.

In some embodiments of the methods the pluripotent stem cell is chosenfrom an embryonic stem (ES) cell, an induced pluripotent stem cell(iPSC), and a cell produced by nuclear transfer. In some embodiments theiPCS cell expresses OCT4, KLF4, SOX2, and cMYC. In some embodiments theMEP co-expresses CD41 and CD235. In some embodiments the MEP does notexpress CD34. In some embodiments the culture begins to make MEP cellswithin 10 days. In some embodiments the culture begins to make MEP cellswithin 7 days. In some embodiments the culture continues to produce newMEP cells for at least 30 days. In some embodiments the number of MEPsproduced in the culture increases exponentially. In some embodiments theculture comprises at least 1 million MEPs per ml. In some embodimentsthe culture comprises at least 10 million MEPs per ml. In someembodiments at least 10% of the cells in the culture are MEPs. In someembodiments at least 50% of the cells in the culture are MEPs. In someembodiments the culture produces at least 10 million MEPs. In someembodiments the culture produces at least 100 million MEPs.

In a related aspect an MEP made by a method of this disclosure is alsoprovided.

In a related aspect a cell culture comprising MEPs made by a method ofthis disclosure is also provided.

In another aspect this disclosure provided methods of making a red bloodcell (RBC). In some embodiments the methods of making a RBC comprisemaking a MEP by a method of this disclosure, and culturing the MEP underconditions sufficient to make a RBC. In some embodiments the conditionssufficient to make a RBC comprise culturing the MEP in the presence ofan AhR agonist. In some embodiments the conditions sufficient to make aRBC comprise culturing in erythroid specification media. In someembodiments the conditions sufficient to make a RBC further compriseculturing in erythroid specification media and culturing in the presenceof an AhR agonist.

In some embodiments the methods of making a RBC comprise providing a MEPthat was made by a method of this disclosure, and culturing the MEPunder conditions sufficient to make a RBC. In some embodiments theconditions sufficient to make a RBC comprise culturing the MEP in thepresence of an AhR agonist. In some embodiments the conditionssufficient to make a RBC comprise culturing in erythroid specificationmedia. In some embodiments the conditions sufficient to make a RBCfurther comprise culturing in erythroid specification media andculturing in the presence of an AhR agonist.

In some embodiments the methods of making a RBC comprise culturing a MEPin the presence of an AhR agonist. The MEP may be from any source. Insome embodiments the methods further comprise culturing the MEP inerythroid specification media.

In some embodiments of the methods, the culture comprises at least 1million RBCs per ml. In some embodiments the culture comprises at least10 million RBCs per ml. In some embodiments at least 10% of the cells inthe culture are RBCs. In some embodiments at least 50% of the cells inthe culture are RBCs. In some embodiments the culture produces at least10 million RBCs. In some embodiments the culture produces at least 100million RBCs.

In a related aspect a RBC made by a method of this disclosure is alsoprovided. Transfusion compositions comprising a RBC made by a method ofthis disclosure are also provided. A culture comprising RBCs made by amethod of this disclosure are also provided.

In another aspect this disclosure provides methods of making amegakaryocyte (Mk). In some embodiments the methods comprise making aMEP by a method of this disclosure, and culturing the MEP underconditions sufficient to make a Mk. In some embodiments the conditionssufficient to make a Mk comprise culturing the MEP in the presence of anAhR modulator. In some embodiments the AhR modulator is an AhRantagonist. In some embodiments the conditions sufficient to make a Mkcomprise culturing the MEP in the presence of megakaryocytespecification media. In some embodiments the conditions sufficient tomake a Mk comprise culturing the MEP in the presence of an AhR modulatorand culturing the MEP in the presence of megakaryocyte specificationmedia. In some embodiments the AhR modulator is an AhR antagonist.

In some embodiments the methods of making a Mk comprise providing a MEPthat was made by a method of this disclosure, and culturing the MEPunder conditions sufficient to make a Mk. In some embodiments theconditions sufficient to make a Mk comprise culturing the MEP in thepresence of an AhR modulator. In some embodiments the AhR modulator isan AhR antagonist. In some embodiments the conditions sufficient to makea Mk comprise culturing the MEP in the presence of megakaryocytespecification media. In some embodiments the conditions sufficient tomake a Mk comprise culturing the MEP in the presence of an AhR modulatorand culturing the MEP in the presence of megakaryocyte specificationmedia. In some embodiments the AhR modulator is an AhR antagonist.

In some embodiments the methods of making an Mk comprise culturing a MEPin the presence of an AhR modulator. In some embodiments the AhRmodulator is an AhR antagonist. The MEP may be from any source. In someembodiments the methods further comprise culturing the MEP inmegakaryocyte specification media.

In a related aspect an Mk made by a method of this disclosure is alsoprovided. A culture comprising Mks made by a method of this disclosureare also provided.

In another aspect this disclosure provides methods of making a platelet.In some embodiments the methods of making a platelet comprise making aMEP by a method of this disclosure, culturing the MEP under conditionssufficient to make a Mk, and culturing the Mk under conditionssufficient for differentiation of a platelet from the Mk. In someembodiments the conditions sufficient to make a Mk comprise culturingthe MEP in the presence of an AhR modulator. In some embodiments the AhRmodulator is an AhR antagonist. In some embodiments the conditionssufficient to make a Mk comprise culturing the MEP in the presence ofmegakaryocyte specification media. In some embodiments the conditionssufficient to make a Mk comprise culturing the MEP in the presence of anAhR modulator and culturing the MEP in the presence of megakaryocytespecification media. In some embodiments the AhR modulator is an AhRantagonist. In some embodiments culturing the Mk under conditionssufficient for differentiation of a platelet from the Mk compriseculturing the Mk in the presence of an AhR modulator. In someembodiments the AhR modulator is an AhR antagonist.

In some embodiments the methods of making a platelet comprise providinga MEP that was made by a method of this disclosure, culturing the MEPunder conditions sufficient to make a Mk, and culturing the Mk underconditions sufficient for differentiation of a platelet from the Mk. Insome embodiments the conditions sufficient to make a Mk compriseculturing the MEP in the presence of an AhR modulator. In someembodiments the AhR modulator is an AhR antagonist. In some embodimentsthe conditions sufficient to make a Mk comprise culturing the MEP in thepresence of megakaryocyte specification media. In some embodiments theconditions sufficient to make a Mk comprise culturing the MEP in thepresence of an AhR modulator and culturing the MEP in the presence ofmegakaryocyte specification media. In some embodiments the AhR modulatoris an AhR antagonist. In some embodiments culturing the Mk underconditions sufficient for differentiation of a platelet from the Mkcomprise culturing the Mk in the presence of an AhR modulator. In someembodiments the AhR modulator is an AhR antagonist.

In some embodiments the methods of making a platelet comprise culturinga MEP in the presence of an AhR modulator. In some embodiments the AhRmodulator is an AhR antagonist. The MEP may be from any source. In someembodiments the methods further comprise culturing the MEP inmegakaryocyte specification media. In some embodiments the methodsfurther comprise culturing the resulting Mk under conditions sufficientfor differentiation of a platelet. In some embodiments culturing the Mkunder conditions sufficient for differentiation of a platelet from theMk comprise culturing the Mk in the presence of an AhR modulator. Insome embodiments the AhR modulator is an AhR antagonist.

In another aspect this disclosure provides methods of differentiating aplatelet from a Mk. In some embodiments the methods comprise culturingthe Mk in the presence of an AhR modulator. In some embodiments the AhRmodulator is an AhR antagonist. In some embodiments the AhR modulatorincreases the rate of propolatelet formation in the culture.

In a related aspect a platelet made by a method of this disclosure isalso provided.

Transfusion compositions comprising a platelet made by a method of thisdisclosure are also provided.

In another aspect this disclosure provides compositions comprising atleast 1 million MEPs per ml. In some embodiments the compositionscomprise at least 10 million MEPs per ml. In some embodiments thecomposition further comprises megakaryocyte erythroid progenitor cells.In some embodiments the composition further comprises RBCs. In someembodiments the composition further comprises megakaryocytes. In someembodiments the composition further comprises platelets.

In another aspect this disclosure provides compositions comprisingcells, wherein at least 10% of the cells are MEPs. In some embodimentsat least 50% of the cells are MEPs. In some embodiments the compositioncomprises at least 1 million MEPs per ml. In some embodiments thecomposition comprises at least 10 million MEPs per ml. In someembodiments the composition further comprises megakaryocyte erythroidprogenitor cells. In some embodiments the composition further comprisesRBCs. In some embodiments the composition further comprisesmegakaryocytes. In some embodiments the composition further comprisesplatelets. In some embodiments the composition is a cell culture.

In another aspect this disclosure provides methods of providing RBCs toa patient in need thereof. In some embodiments the methods comprisetransfusing a composition comprising RBCs made by a method of thisdisclosure into the circulatory system of the patient.

In another aspect this disclosure provides methods of treating anemia ina patient in need thereof. In some embodiments the methods comprisetransfusing a composition comprising RBCs made by a method of thisdisclosure into the circulatory system of the patient. In someembodiments the anemia is caused by at least one of impaired productionof RBCs, increased destruction of RBCs, blood loss, and fluid overload.In some embodiments the anemia is caused by thalassemia. In someembodiments the anemia is sickle cell anemia. In some embodiments theRBCs are blood type matched to the patient. In some embodiments the RBCsare differentiated from pluripotent stem cells isolated from thepatient.

In another aspect this disclosure provides methods of providingplatelets to a patient in need thereof. In some embodiments the methodscomprise transfusing a composition comprising platelets made by a methodof this disclosure into the circulatory system of the patient.

In another aspect this disclosure provides methods of treatingthrombocytopenia in a patient in need thereof. In some embodiments themethods comprise transfusing a composition comprising platelets made bya method of this disclosure into the circulatory system of the patient.In some embodiments the thrombocytopenia is caused by at least one ofdecreased production of platelets, increased destruction of platelets,and a medication. In some embodiments the platelets are blood typematched to the patient. In some embodiments the platelets aredifferentiated from pluripotent stem cells isolated from the patient.

In another aspect this disclosure provides methods of screening acompound for an effect on a RBC. In some embodiments the methodscomprise a) making a RBC by a method of this disclosure, b) contactingthe RBC with the compound, and c) observing a change in the RBC.

In another aspect this disclosure provides alternative methods ofscreening a compound for an effect on a RBC. The methods comprise a)providing a RBC that was made by a method of this disclosure, b)contacting the RBC with the compound, and c) observing a change in theRBC.

In another aspect this disclosure provides methods of screening acompound for an effect on a Mk. In some embodiments the methods comprisea) making a Mk by a method of this disclosure, b) contacting the Mk withthe compound, and c) observing a change in the Mk.

In another aspect this disclosure provides alternative methods ofscreening a compound for an effect on a Mk. In some embodiments themethods comprise a) providing a Mk that was made by a method of thisdisclosure, b) contacting the Mk with the compound, and c) observing achange in the Mk.

In another aspect this disclosure provides methods of screening acompound for an effect on a platelet. In some embodiments the methodscomprise a) making a platelet by a method of this disclosure, b)contacting the platelet with the compound, and c) observing a change inthe platelet.

In another aspect this disclosure provides alternative methods ofscreening a compound for an effect on a platelet. In some embodimentsthe methods comprise providing a platelet that was made by a method ofthis disclosure, b) contacting the platelet with the compound, and c)observing a change in the platelet.

In another aspect this disclosure provides methods of increasing theplatelet count of a mammal. In some embodiments the methods compriseadministering an effective amount of an AhR agonist to the mammal.

In another aspect this disclosure provides methods of treatingthrombocytopenia in a mammal. In some embodiments the methods compriseadministering an effective amount of an AhR agonist to the mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A to 1B show an analysis of human hematopoietic celldifferentiation genomic mapping (dMap) data. A computational analysis ofcomprehensive microarray data obtained through the Broad Institute'sDifferential Map Portal (dMAP) was performed. The genes were sortedbased on hierarchical clustering with 1-Pearson correlation as thedistance metric, and average linkage as the agglomeration rule (1A). Thenormalized expression level of AhR within each cell population(sub-population) was computed and visualized by means ofbox-and-whiskers plots (1B). For each population, the plot reports themedian (thick mid line), the middle half (the box), and theInterquartile Range (IQR, the distance between the “whiskers”) of thedistribution of AhR values. The difference in the expression level ofAhR among cell populations was tested by standard analysis-of-variance(anova).

FIGS. 2A to 2D show that the feeder-free, chemically defined productionof megakaryocyte-erythroid progenitors (MEPs) from induced pluripotentstem cells (iPSCs) produces populations of cells that express definitivemarkers of both the megakaryocyte and erythroid lineages. (A)Differentiation strategy from iPSC to MEP stage. Phase contrast imagesof culture depicting morphological changes and the production of both aninitial adherent layer followed by non-adherent MEPs. (B) RepresentativeFACS analysis of Day 13 MEPs that co-express CD235-PE (red cells) andCD41-FITC (megakaryocytes). (C) FACS analysis of Day 13 MEPs that havebeen exposed to either erythroid or megakaryocyte-specific specificationmedia for 5 days. (D) qPCR analysis of undifferentiated iPSCs vs. Day 13MEPs. Relative gene expression was normalized to β-actin. Data isaverage of triplicate wells+SD. *p<0.05.

FIGS. 3A to 3D show that the aryl hydrocarbon receptor (AhR) agonistFICZ inhibits apoptosis and allows for the exponential expansion ofiPSC-derived MEPs: (A) Representative FACS dot plots of live versus deadcells (PI vs. Hoechst) from D15 MEPs+FICZ. Plots were gated first in FSCvs. SSC and then from that population for PI⁺ and PI⁻ Hoechst⁺. FICZincreases the population of live cells as delineated by FSC and SSC(32.6%) as well as PI⁻ Hoechst⁻ (97.7%). (B) Representative phasecontrast images of MEP population+FICZ. (C) Growth curve of D15 MEPs+/−0.2 μm FICZ. Cells were counted manually using trypan blue exclusion.Graphical data and the associated statistics are the result of threeindependent experiments per group. (D) Day 30 MEPs that have beentreated with the AhR agonist FICZ are more proliferative than untreatedcells as quantified by EDU incorporation.

FIGS. 4A and 4B show that AhR agonists induce CYP1B1 target geneexpression in human iPSCs and MEPs. (A) Western blot analysis for AhRand β-actin protein expression in iPSC and MEPs. (B) qPCR data of iPSCand Day 15 MEPs with and without FICZ. Expression is normalized toβ-actin levels. Data is average of triplicate wells+SD. *p<0.05,**p<0.005.

FIGS. 5A to 5F show that AhR mediates the expansion and specification ofbipotential hematopoietic progenitors. (A) Schematic representation ofpHAGE2 lentiviral reporter constructs that contain the mouse mammarytumor virus flanking dioxin response element region from the murineCY1A1 gene (MMTV-DRE-MMTV) driving the expression of NLS-dsRed orluciferase IRES zsGreen (pHAGE2-MMTV-DRE-MMTV-NLS-dsRed-IRES-zsGreen andpHAGE2-MMTV-DRE-MMTV-luciferase-IRES-zsGreen). (B) FACS analysis forNLS-dsRED in MEPs infected withpHAGE2-MMTV-DRE-MMTV-NLS-dsRed-IRES-zsGreen. Infected cells wereuntreated or treated with 5 μM CH223191, or 0.404 FICZ. (C) Relativefluorescence units of cells infected with luciferase vector with orwithout FICZ or CH223191. (D) Phase contrast and fluorescent images ofzs-Green expression in mock infected or infected cells. (E)Representative flow cytometry dot plots of live versus dead cells (PIvs. Hoechst) from D13 MEPs+FICZ and/or CH223191. For these experiments,MEPs were pretreated with the known AhR inhibitor CH223191 at D6 beforethe addition of FICZ at D7. (F) qPCR results of MEPs from “E”,normalized to β-actin. Data is average of triplicate wells+SD. *p<0.005.

FIGS. 6A to 6J show continuous AhR activation allows for red cellmaturation while inhibition/antagonism promotes megakaryocytedevelopment/specification. (A) Representative FACS analysis dot plots ofcells co-expressing CD235-PE and CD71-FITC over time. (B) RepresentativeFACS analysis dot plots of cells co-expressing CD235-PE and CD41-FITC.(C) Wright-Giemsa stain of immature and mature MEPs. (D) Hemoglobinexpressing cell pellets of MEPs±EPO. (E) Representative FACS analysisdot plots of cells co-expressing CD235-PE and CD41-FITC±CH223191. (F)Schematic representation of pHAGE2 lentiviral reporter constructcontaining the AhR repressor (AHRR) and zsGreen under control of theconstitutive promoter Eflα (pHAGE2-Eflα-AHRR-IRES-zsGreen). (G)Representative FACS dot plots of cells infected with mock orpHAGE2-Eflα-AHRR-IRES-zsGreen showing CD235-PE or CD41-PE expression.(H) Wright-Giemsa stain of megakaryocytes produced by AhR antagonism.(I) Ploidy analysis by FACS of the produced megakaryocytes. (J) Phaseand fluorescent images of the large cells (megakaryocytes) expressing azsGreen reporter that marks cells co-expressing the AhRR element.

FIG. 7 shows a mechanistic diagram of AhR involvement in nominalhematopoietic development. AhR agonism allows for the production andexpansion of a megakaryocyte erythroid progenitor (MEP) population.Continued AhR agonism is permissive for red cell development whereas AhRantagonism preferentially directs the MEPs to become megakaryocytes.

FIGS. 8A and 8B show the expression of genes involved in thereprogramming of iPSCs and the genes involved in RBC differentiation.(A) embryonic genes (including those such as Oct4, Sox2, and Nanog thatare responsible for the reprogramming process are downregulated as cellsare directly differentiated into RBCs. (B) At days 15 and 30 oferythroid specification in this directed differentiation system thecells exhibit a complementary heavy upregulation of genes of criticalimport to RBCs.

FIGS. 9A and 9B show mass spectrophotometric analyses of globin geneexpression in human whole blood. Analyses of whole peripheral blood of acontrol patient (A) and a patient suffering from sickle cell disease(9B) is shown.

FIG. 10 shows mass spectrophotometric analyses of globin gene expressionin iPSC-derived RBCs made by methods of this disclosure.

FIG. 11 shows that exposure of iPSC-derived RBCs to 0.5 μM hydroxyureaHU causes an approimately 4-fold increase in expression of fetalhemoglobin (HbF; gamma) indicating that iPSC-derived RBCs are responsiveto HbF inducers.

FIG. 12 shows that AhR agonism promotes MEP production and expansion inmurine bone marrow. Representative FACS analysis dot plots of red celldepleted C57B6 bone marrow grown for 3 days in +/−0.2 μM FICZ. 1×10̂5cells were initially treated with CD16/32 Fc receptor block, followed bydirectly conjugated monoclonal antibodies for the designated markers.

FIGS. 13A and 13B show that iPSCs and MEPs are responsive to a spectrumof AhR agonists. (A) RT-PCR analysis of CYP1B1 in iPSC treated with TCDDor β-NF for 4 days. Data are averages of duplicate wells +SE and valuesare normalized to GAPDH. (B) RT-PCR analysis of CYP1B1 in MEP treatedwith β-NF or FICZ. Data are averages of duplicate wells+SE and valuesnormalized to GAPDH.

FIG. 14 shows that iPSC-Mks, created using AhR antagonism, express aseries of hallmark and characteristic MK markers.

FIG. 15 shows that iPSC-derived platelets are remarkably similar tothose derived from whole blood.

FIGS. 16A to 16C show that AhR agonist FICZ is active in vivo andresults in increased platelet counts in normal mice. (A) C57B6 mice wereinjected daily intraperitoneally with FICZ suspended in vegetable oilusing a weekly dose escalation scheme (Week 1: 1 mg/kg; Week 2: 2 mg/kg;Week 3: 4 mg/kg). Hemavet quantification of peripheral blood bleeds weredone at 3 time points (Day 7, 14, and 21) Interestingly, a mouse thatwas immediately exposed to higher doses of FICZ and did not undergo week1 escalation demonstrated a more immediate and prolific plateletresponse. (B) Following the 3 week time point, mice were sacrificed andtheir livers were harvested for quantitative RT-PCR analysis for CYP 1B1target gene expression. (C) Following the 3 week time point, mice weresacrificed and their spleens were harvested for quantitative RT-PCRanalysis for CYP1B1 target gene expression.

FIG. 17 shows a short hairpin RNA (RNAi) for AhR construct (bottom)which can be turned on in undifferentited and differentiating iPSCs(top).

FIG. 18 shows that activation of the construct in Mks causes a dramaticincrease in proplatelet formation.

DETAILED DESCRIPTION

Unless otherwise defined herein, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall include theplural and plural terms shall include the singular. Generally,nomenclatures used in connection with, and techniques of, biochemistry,enzymology, molecular and cellular biology, microbiology, genetics andprotein and nucleic acid chemistry and hybridization described hereinare those well-known and commonly used in the art. Certain referencesand other documents cited herein are expressly incorporated herein byreference. Additionally, all Genbank or other sequence database recordscited herein are hereby incorporated herein by reference. In case ofconflict, the present specification, including definitions, willcontrol. The materials, methods, and examples are illustrative only andnot intended to be limiting.

The methods and techniques of the present disclosure are generallyperformed according to conventional methods well known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification unless otherwiseindicated. See, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (2001); Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Associates (1992, and Supplements to 2002); Taylor andDrickamer, Introduction to Glycobiology, Oxford Univ. Press (2003);Worthington Enzyme Manual, Worthington Biochemical Corp., Freehold,N.J.; Handbook of Biochemistry: Section A Proteins, Vol I, CRC Press(1976); Handbook of Biochemistry: Section A Proteins, Vol II, CRC Press(1976); Essentials of Glycobiology, Cold Spring Harbor Laboratory Press(1999).

This disclosure refers to sequence database entries (e.g., Genbank andUniProt records) for certain amino acid and nucleic acid sequences thatare published on the internet, as well as other information on theinternet. The skilled artisan understands that information on theinternet, including sequence database entries, is updated from time totime and that, for example, the reference number used to refer to aparticular sequence can change. Where reference is made to a publicdatabase of sequence information or other information on the internet,it is understood that such changes can occur and particular embodimentsof information on the internet can come and go. Because the skilledartisan can find equivalent information by searching on the internet, areference to an internet web page address or a sequence database entryevidences the availability and public dissemination of the informationin question.

Before the present compositions, methods, and other embodiments aredisclosed and described, it is to be understood that the terminologyused herein is for the purpose of describing particular embodiments onlyand is not intended to be limiting. It must be noted that, as used inthe specification and the appended claims, the singular forms “a,” “an”and “the” include plural referents unless the context clearly dictatesotherwise.

The term “comprising” as used herein is synonymous with “including” or“containing”, and is inclusive or open-ended and does not excludeadditional, unrecited members, elements or method steps.

As used herein, the term “in vitro” refers to events that occur in anartificial environment, e.g., in a test tube or reaction vessel, in cellculture, in a Petri dish, etc., rather than within an organism (e.g.,animal, plant, or microbe).

As used herein, the term “in vivo” refers to events that occur within anorganism (e.g., animal, plant, or microbe).

As used herein, the term “isolated” refers to a substance or entity thathas been (1) separated from at least some of the components with whichit was associated when initially produced (whether in nature or in anexperimental setting), and/or (2) produced, prepared, and/ormanufactured by the hand of man. Isolated substances and/or entities maybe separated from at least about 10%, about 20%, about 30%, about 40%,about 50%, about 60%, about 70%, about 80%, about 90%, or more of theother components with which they were initially associated. In someembodiments, isolated agents are more than about 80%, about 85%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, about 99%, or more than about 99% pure. As usedherein, a substance is “pure” if it is substantially free of othercomponents.

The MEPs, RBCs, megakaryocytes, and platelets of this disclosure aretypically mammalian or marsupial cells. As used herein “mammal” and“mammalian” refers to any member of the taxonomic class mammal,including without limitation, all primates including humans; rodents,including mice and rats; farm animals including pigs, horses, cattle,sheep, and goats; and companion animals including dogs and cats.

The term “peptide” as used herein refers to a short polypeptide, e.g.,one that typically contains less than about 50 amino acids and moretypically less than about 30 amino acids. The term as used hereinencompasses analogs and mimetics that mimic structural and thusbiological function.

The term “polypeptide” encompasses both naturally-occurring andnon-naturally occurring proteins, and fragments, mutants, derivativesand analogs thereof. A polypeptide may be monomeric or polymeric.Further, a polypeptide may comprise a number of different domains eachof which has one or more distinct activities. For the avoidance ofdoubt, a “polypeptide” may be any length greater two amino acids.

The term “isolated protein” or “isolated polypeptide” is a protein orpolypeptide that by virtue of its origin or source of derivation (1) isnot associated with naturally associated components that accompany it inits native state, (2) exists in a purity not found in nature, wherepurity can be adjudged with respect to the presence of other cellularmaterial (e.g., is free of other proteins from the same species) (3) isexpressed by a cell from a different species, or (4) does not occur innature (e.g., it is a fragment of a polypeptide found in nature or itincludes amino acid analogs or derivatives not found in nature orlinkages other than standard peptide bonds). Thus, a polypeptide that ischemically synthesized or synthesized in a cellular system differentfrom the cell from which it naturally originates will be “isolated” fromits naturally associated components. A polypeptide or protein may alsobe rendered substantially free of naturally associated components byisolation, using protein purification techniques well known in the art.As thus defined, “isolated” does not necessarily require that theprotein, polypeptide, peptide or oligopeptide so described has beenphysically removed from a cell in which it was synthesized.

The term “polypeptide fragment” as used herein refers to a polypeptidethat has a deletion, e.g., an amino-terminal and/or carboxy-terminaldeletion compared to a full-length polypeptide, such as a naturallyoccurring protein. In an embodiment, the polypeptide fragment is acontiguous sequence in which the amino acid sequence of the fragment isidentical to the corresponding positions in the naturally-occurringsequence. Fragments typically are at least 5, 6, 7, 8, 9 or 10 aminoacids long, or at least 12, 14, 16 or 18 amino acids long, or at least20 amino acids long, or at least 25, 30, 35, 40 or 45, amino acids, orat least 50 or 60 amino acids long, or at least 70 amino acids long.

The term “fusion protein” refers to a polypeptide comprising apolypeptide or fragment coupled to heterologous amino acid sequences.Fusion proteins are useful because they can be constructed to containtwo or more desired functional elements that can be from two or moredifferent proteins. A fusion protein comprises at least 10 contiguousamino acids from a polypeptide of interest, or at least 20 or 30 aminoacids, or at least 40, 50 or 60 amino acids, or at least 75, 100 or 125amino acids. The heterologous polypeptide included within the fusionprotein is usually at least 6 amino acids in length, or at least 8 aminoacids in length, or at least 15, 20, or 25 amino acids in length.Fusions that include larger polypeptides, such as an IgG Fc region, andeven entire proteins, such as the green fluorescent protein (“GFP”)chromophore-containing proteins, have particular utility. Fusionproteins can be produced recombinantly by constructing a nucleic acidsequence which encodes the polypeptide or a fragment thereof in framewith a nucleic acid sequence encoding a different protein or peptide andthen expressing the fusion protein. Alternatively, a fusion protein canbe produced chemically by crosslinking the polypeptide or a fragmentthereof to another protein.

As used herein, a protein has “homology” or is “homologous” to a secondprotein if the nucleic acid sequence that encodes the protein has asimilar sequence to the nucleic acid sequence that encodes the secondprotein. Alternatively, a protein has homology to a second protein ifthe two proteins have similar amino acid sequences. (Thus, the term“homologous proteins” is defined to mean that the two proteins havesimilar amino acid sequences.) As used herein, homology between tworegions of amino acid sequence (especially with respect to predictedstructural similarities) is interpreted as implying similarity infunction.

When “homologous” is used in reference to proteins or peptides, it isrecognized that residue positions that are not identical often differ byconservative amino acid substitutions. A “conservative amino acidsubstitution” is one in which an amino acid residue is substituted byanother amino acid residue having a side chain (R group) with similarchemical properties (e.g., charge or hydrophobicity). In general, aconservative amino acid substitution will not substantially change thefunctional properties of a protein. In cases where two or more aminoacid sequences differ from each other by conservative substitutions, thepercent sequence identity or degree of homology may be adjusted upwardsto correct for the conservative nature of the substitution. Means formaking this adjustment are well known to those of skill in the art. See,e.g., Pearson, 1994, Methods Mol. Biol. 24:307-31 and 25:365-89.

The following six groups each contain amino acids that are conservativesubstitutions for one another: 1) Serine, Threonine; 2) Aspartic Acid,Glutamic Acid; 3) Asparagine, Glutamine; 4) Arginine, Lysine; 5)Isoleucine, Leucine, Methionine, Alanine, Valine, and 6) Phenylalanine,Tyrosine, Tryptophan.

Sequence homology for polypeptides, which is also referred to as percentsequence identity, is typically measured using sequence analysissoftware. See, e.g., the

Sequence Analysis Software Package of the Genetics Computer Group (GCG),University of Wisconsin Biotechnology Center, 910 University Avenue,Madison, Wis. 53705. Protein analysis software matches similar sequencesusing a measure of homology assigned to various substitutions, deletionsand other modifications, including conservative amino acidsubstitutions. For instance, GCG contains programs such as “Gap” and“Bestfit” which can be used with default parameters to determinesequence homology or sequence identity between closely relatedpolypeptides, such as homologous polypeptides from different species oforganisms or between a wild-type protein and a mutein thereof. See,e.g., GCG Version 6.1.

An exemplary algorithm when comparing a particular polypeptide sequenceto a database containing a large number of sequences from differentorganisms is the computer program BLAST (Altschul et al., J. Mol. Biol.215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993);Madden et al., Meth. Enzymol. 266:131-141 (1996); Altschul et al.,Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res.7:649-656 (1997)), especially blastp or tblastn (Altschul et al.,Nucleic Acids Res. 25:3389-3402 (1997)).

Exemplary parameters for BLASTp are: Expectation value: 10 (default);Filter: seg (default); Cost to open a gap: 11 (default); Cost to extenda gap: 1 (default); Max. alignments: 100 (default); Word size: 11(default); No. of descriptions: 100 (default); Penalty Matrix:BLOWSUM62. The length of polypeptide sequences compared for homologywill generally be at least about 16 amino acid residues, or at leastabout 20 residues, or at least about 24 residues, or at least about 28residues, or more than about 35 residues. When searching a databasecontaining sequences from a large number of different organisms, it maybe useful to compare amino acid sequences. Database searching usingamino acid sequences can be measured by algorithms other than blastpknown in the art. For instance, polypeptide sequences can be comparedusing FASTA, a program in GCG Version 6.1. FASTA provides alignments andpercent sequence identity of the regions of the best overlap between thequery and search sequences. Pearson, Methods Enzymol. 183:63-98 (1990).For example, percent sequence identity between amino acid sequences canbe determined using FASTA with its default parameters (a word size of 2and the PAM250 scoring matrix), as provided in GCG Version 6.1, hereinincorporated by reference.

In some embodiments, polymeric molecules (e.g., a polypeptide sequenceor nucleic acid sequence) are considered to be “homologous” to oneanother if their sequences are at least 25%, at least 30%, at least 35%,at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 99% identical. In some embodiments,polymeric molecules are considered to be “homologous” to one another iftheir sequences are at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, or at least 99% similar. The term “homologous”necessarily refers to a comparison between at least two sequences(nucleotides sequences or amino acid sequences). In some embodiments,two nucleotide sequences are considered to be homologous if thepolypeptides they encode are at least about 50% identical, at leastabout 60% identical, at least about 70% identical, at least about 80%identical, or at least about 90% identical for at least one stretch ofat least about 20 amino acids. In some embodiments, homologousnucleotide sequences are characterized by the ability to encode astretch of at least 4-5 uniquely specified amino acids. Both theidentity and the approximate spacing of these amino acids relative toone another must be considered for nucleotide sequences to be consideredhomologous. In some embodiments of nucleotide sequences less than 60nucleotides in length, homology is determined by the ability to encode astretch of at least 4-5 uniquely specified amino acids. In someembodiments, two protein sequences are considered to be homologous ifthe proteins are at least about 50% identical, at least about 60%identical, at least about 70% identical, at least about 80% identical,or at least about 90% identical for at least one stretch of at leastabout 20 amino acids.

As used herein, a “modified derivative” refers to polypeptides orfragments thereof that are substantially homologous in primarystructural sequence to a reference polypeptide sequence but whichinclude, e.g., in vivo or in vitro chemical and biochemicalmodifications or which incorporate amino acids that are not found in thereference polypeptide. Such modifications include, for example,acetylation, carboxylation, phosphorylation, glycosylation,ubiquitination, labeling, e.g., with radionuclides, and variousenzymatic modifications, as will be readily appreciated by those skilledin the art. A variety of methods for labeling polypeptides and ofsubstituents or labels useful for such purposes are well known in theart, and include radioactive isotopes such as ¹²⁵1, ³²P, ³⁵S, and ³H,ligands that bind to labeled antiligands (e.g., antibodies),fluorophores, chemiluminescent agents, enzymes, and antiligands that canserve as specific binding pair members for a labeled ligand. The choiceof label depends on the sensitivity required, ease of conjugation withthe primer, stability requirements, and available instrumentation.Methods for labeling polypeptides are well known in the art. See, e.g.,Ausubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates (1992, and Supplements to 2002).

As used herein, “polypeptide mutant” or “mutein” refers to a polypeptidewhose sequence contains an insertion, duplication, deletion,rearrangement or substitution of one or more amino acids compared to theamino acid sequence of a reference protein or polypeptide, such as anative or wild-type protein. A mutein may have one or more amino acidpoint substitutions, in which a single amino acid at a position has beenchanged to another amino acid, one or more insertions and/or deletions,in which one or more amino acids are inserted or deleted, respectively,in the sequence of the reference protein, and/or truncations of theamino acid sequence at either or both the amino or carboxy termini. Amutein may have the same or a different biological activity compared tothe reference protein.

In some embodiments, a mutein has, for example, at least 85% overallsequence homology to its counterpart reference protein. In someembodiments, a mutein has at least 90% overall sequence homology to thewild-type protein. In other embodiments, a mutein exhibits at least 95%sequence identity, or 98%, or 99%, or 99.5% or 99.9% overall sequenceidentity.

As used herein, the term “agonist” refers to an agent that triggers aresponse that is at least one response triggered by binding of anendogenous ligand of a receptor to the receptor. In some embodiments,the agonist may act directly or indirectly on a second agent that itselfmodulates the activity of the receptor. In some embodiments, the atleast one response of the receptor is an activity of the receptor thatcan be measured with assays including but not limited to physiological,pharmacological, and biochemical assays. Exemplary assays include butare not limited to assays that measure the binding of an agent to thereceptor, the binding of the receptor to a substrate such as but notlimited to a nuclear receptor and a regulatory element of a target gene,the effect on gene expression assayed at the mRNA or resultant proteinlevel, and the effect on an activity of proteins regulated eitherdirectly or indirectly by the receptor. For example, AhR receptoractivity may be measures by monitoring the expression of an AhR-targetgene, such as CYP1B1.

As used herein, the term “antagonist” refers to an agent that inhibits aresponse that is at least one response triggered by binding of anagonist of a receptor to the receptor. In some embodiments, theantagonist may act directly or indirectly on a second agent that itselfmodulates the activity of the receptor. In some embodiments, the atleast one response of the receptor is an activity of the receptor thatcan be measured with assays including but not limited to physiological,pharmacological, and biochemical assays. Exemplary assays include butare not limited to assays that measure the binding of an agent to thereceptor, the binding of the receptor to a substrate such as but notlimited to a nuclear receptor and a regulatory element of a target gene,the effect on gene expression assayed at the mRNA or resultant proteinlevel, and the effect on an activity of proteins regulated eitherdirectly or indirectly by the receptor. For example, AhR receptoractivity may be measures by monitoring the expression of an AhR-targetgene, such as CYP1B1.

As used herein, the term “agent” or “active agent” refers to a substanceincluding, but not limited to a chemical compound, such as a smallmolecule or a complex organic compound, a protein, such as an antibodyor antibody fragment or a protein comprising an antibody fragment, or agenetic construct which acts at the DNA or mRNA level in an organism.

As used herein, the term “modulating” and “modulate” refers to changingor altering an activity, function, or feature. The term “modulator”refers to an agent which modulates an activity, function, or feature.For example, an agent may modulate an activity by increasing ordecreasing the activity compared to the effects on the activity in theabsence of the agent. In some embodiments, a modulator that increases anactivity, function, or feature is an agonist. In some embodiments, amodulator that increases an activity, function, or feature is anantagonist.

As used herein, the terms “treat,” “treatment,” “treating,” and“amelioration” refer to therapeutic treatments, wherein the object is toreverse, alleviate, ameliorate, inhibit, slow down and/or stop theprogression or severity of a condition associated with a disease ordisorder. The terms include reducing or alleviating at least one adverseeffect or symptom of a condition, disease or disorder associated with adeficiency in the number or defect in the quality of at least one bloodcell type, such as platelets. Treatment is generally “effective” if oneor more symptoms or clinical markers are reduced. Alternatively,treatment is “effective” if the progression of a disease is reduced orhalted. That is, “treatment” includes not just the improvement ofsymptoms or markers, but also a cessation of at least slowing ofprogress or worsening of symptoms that would be expected in absence oftreatment. Beneficial or desired clinical results include, but are notlimited to, alleviation of one or more symptom(s), diminishment ofextent of disease, stabilized (i.e., not worsening) state of disease,delay or slowing of disease progression, amelioration or palliation ofthe disease state, and remission (whether partial or total), whetherdetectable or undetectable. The terms “treat,” “treatment,” “treating,”and “amelioration” in reference to a disease also include providingrelief from the symptoms or side-effects of the disease (includingpalliative treatment).

As used herein, “co-administred” and “co-administration” refer toadministration of at least two agents to a mammal to treat a condition,wherein the at least two agents are administered for therapeutic dosingperiods that overlap for administration of at least one does of eachagent. For example, if agent A is administered on day 1, agent B isadministered on day 2, and agent A is administered on day 3 then agentsA and B are co-administered. Therapeutic dosing periods may comprise 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 or more administrations of an agent.Administration may be daily, three times a week, two times a week,weekly, every two weeks, or monthly, for example.

A. Introduction to the Disclosure

The differentiation of HSCs into all eight blood cell lineages is atightly regulated and critical physiological process that changes insubtle but important ways during the lifespan of the individual.Disruption of this regulation can have a profound downstream effect onmultiple hematopoietic cell types, potentially leading tomyelodysplasia, mixed lineage leukemias, CML, lymphomas, stem cellexhaustion, thrombocytopenia, anemia and other blood cell disorders.However, definition of the molecular mechanisms that controlspecification of primary human blood cells has been hampered by a lackof platforms with which sufficient numbers of stem or progenitor cellscan be grown and the absence of practical and efficient techniques fordirecting differentiation of those cells into end stage cells. Forexample, several teams have published proof-of-principle examples of thederivation of megakaryocytes (Mks) (1) and erythroid-lineage cells (2)from embryonic stem cells (ESC) and induced pluripotent stem cells(iPSC). However, development of a model system which results in robustexpansion of these cell populations and with which molecular signalsdriving cell differentiation can readily be studied has beenproblematic.

Our conceptual approach to addressing this glaring unmet need has beento mimic the natural sequences of hematopoietic cell development invitro to derive the number and range of cells types needed for thecreation of a genetically tractable iPSC-based platform. A key componentof this new platform, as shown here, is the demonstration that arylhydrocarbon receptor (AhR) hyper-activation enables outgrowth ofmyeloid-erythroid progenitor cells and production of Mk anderythroid-lineage cells from iPSCs.

The AhR is a member of the evolutionarily conserved Per/ARNT/SIM (PAS)family of transcription factors. It is the only PAS family member knownto be activated by endogenous or exogenous ligands. PAS proteinscontribute to several important physiological processes. Historically,the evolutionarily conserved AhR was studied in the context of itsactivation by a variety of ubiquitous environmental pollutants includingdioxins, polychlorinated biphenyls, and polycyclic aromatichydrocarbons, and subsequent transactivation of cytochrome P450-encodinggenes, the products of which catalyze production of mutagenic or toxicintermediates. However, the AhR field has recently undergone a majorparadigm shift following the demonstration that the AhR plays importantphysiological roles in the absence of environmental ligands. Forexample, several studies demonstrate that the AhR contributes toregulation of autoimmune responses, inflammation, cell growth, cellmigration, apoptosis and cancer progression. Specifically with regard tohematopoietic cells, several high profile studies demonstrate that theAhR regulates development of Th17 cells, regulatory T cells subsets, andgut-associated T cells.

Importantly, recent breakthrough studies suggest that the AhR plays acritical role in nominal HSC growth and differentiation. For example,AhR−/− mice are characterized by an increased number of bone marrow HSCsand a commensurate increased propensity to develop lymphomas.Furthermore, AhR−/− mice produce decreased numbers of erythrocytes andplatelets, lower-ploidy Mks, and increased numbers of B lineage andmyeloid cells. These results led to the hypothesis that the AhR,activated by endogenous ligands, regulates stem cell growth and/ordifferentiation.

Despite these early results, many important questions remain.Specifically, little is known of the effects of AhR modulation on thedevelopment of Mk or erythroid-lineage cells from bipotentialprogenitors. That the AhR is involved in this process is suggested bydecreased numbers of HSCs, erythrocytes and platelets in young AhR−/−mice and the skewing of the blood cell repertoire towards myeloid and Blineage cells as AhR−/− mice age.

To build on these studies and to develop a robust system for studying Mkand erythroid cell differentiation, we developed a novel, feeder-freeand chemically-defined protocol for the directed differentiation ofiPSCs into hematopoietic progenitor cells and their progeny. A necessarycomponent of this system was shown to be the hyper-activation of the AhRwith a potent AhR agonist, 6-formylindole(3,2-b)carbazole (FICZ). The invitro system described herein allows, in some embodiments, the capturein culture and expansion of pure populations of megakaryocyte-erythroidprogenitors that exist transiently during in vivo development in theproduction of end stage red blood cells (RBCs) and Mks. This platform insome embodiments allows for unprecedented efficiency and consistency inthe derivation of bi-potential hematopoietic progenitors and progenyproduction from pluripotent stem cells using AhR modulation. In additionto demonstrating a critical role for the AhR in MEP, Mk, and RBCdevelopment, the platform provides an important and geneticallytractable system for studying blood cell differentiation at multiple,defined stages of development. Perhaps most importantly, the platformpresented here represents a significant step forward towards the invitro production of therapeutic, patient-specific platelets and RBC.

Furthermore, this work indicates that AhR has a physiological andfunctional role in hematopoiesis, and that modulation of the receptor inbi-potential hematopoietic progenitors can direct cell fate.

B. Stem Cells

Stem cells are cells in multicellular organisms that can divide anddifferentiate into diverse specialized cell types and can self-renew toproduce more stem cells that have the same property. A “pluripotent stemcell” as used herein is a stem cell that has the potential todifferentiate into any of the three germ layers: endoderm (e.g.,interior stomach lining, gastrointestinal tract, the lungs), mesoderm(e.g., muscle, bone, blood, urogenital system), and ectoderm (e.g.,epidermal tissues and nervous system). Pluripotent stem cells can giverise to any fetal or adult cell type. For the purposes of thisdisclosure a “pluripotent stem cell” may include a totipotent stem cell,which is a cell that can construct a complete, viable organism. Thesecells are produced from the fusion of an egg and sperm cell. Cellsproduced by the first few divisions of the fertilized egg are alsototipotent. Pluripotent stem cells include but are not limited toembryonic stem (ES) cells, induced pluripotent stem cells (iPSC), andcells produced by somatic cell nuclear transfer (SCNT).

ES cells are totipotent stem cells derived from the inner cell mass ofthe blastocyst of an early-stage mammalian embryo. Methods of derivingmammalian ES cells are well known in the art as are numerous establishedES cell lines that may be used in conjunction with certain embodimentsof this disclosure.

iPSCs are a type of pluripotent stem cell artificially derived from anon-pluripotent cell—typically an adult somatic cell—by inducing the“forced” expression of specific genes. Induced pluripotent stem cellsare similar to natural pluripotent stem cells, such as embryonic stem(ES) cells, in many aspects, such as, in some embodiments, at least oneof the expression of certain stem cell genes and proteins, chromatinmethylation patterns, doubling time, embryoid body formation, teratomaformation, viable chimera formation, and potency and differentiability,but the full extent of their relation to natural pluripotent stem cellsis still being assessed.

iPSCs are typically derived by transfection of certain stemcell-associated genes into non-pluripotent cells, such as adultfibroblasts. Transfection is typically achieved through viral vectors,such as retroviruses. Transfected genes may include the mastertranscriptional regulators Oct-3/4 (Pou5f1) and Sox2. Over timefollowing transfection small numbers of transfected cells begin tobecome morphologically and biochemically similar to pluripotent stemcells, and are typically isolated through at least one of morphologicalselection, doubling time, a reporter gene and antibiotic selection.

In some embodiments the iPSC is formed by a method comprisingtransfecting a somatic cell with open reading frames that encode theOct-3/4, SOX2, c-Myc, and Klf4 proteins. In some embodiments the iPSC isformed by a method comprising transfecting a somatic cell with openreading frames that encode the OCT4, SOX2, NANOG, and LIN28 proteins. Insome embodiments the transfection comprises introducing a retroviralvector into the somatic cell. In alternative embodiments, the iPSC isformed by a method comprising treating the somatic cell with at leastone small molecule inducer of iPSC formation. In some embodiments theiPSC is formed by a method comprising treating the somatic cell with atleast one small molecule inducer of iPSC formation and transfecting thesomatic cell with open reading frames that encodes a protein inducer ofiPSC formation. In such embodiments the at least one protein may beselected from Oct-3/4, SOX2, c-Myc, Klf4, NANOG, and LIN28.

iPSCs can give rise to multipotent stem cells. In the hematopoieticlineage an iPSC or ES cell can give rise to a cell in a hemangioblasticstate. The hemangioblastic cell then in turn gives rise to ahematopoietic stem cell which gives rise to MEP cells.

As will be apparent to a skilled artisan reading this disclosure, anypluripotent stem cell or any multipotent stem cell capable ofdifferentiating into a MEP may be used in embodiments of the methodsdisclosed herein to make RBCs and/or platelets.

C. Hematopoietic Cell Types

All cellular blood components are derived from hematopoietic stem cells(HSCs). In a healthy adult person, approximately 10¹¹-10¹² new bloodcells are produced daily in order to maintain steady state levels in theperipheral circulation. HSCs reside in the medulla of the bone (bonemarrow) and have the unique ability to give rise to all of the differentmature blood cell types. HSCs are self-renewing: when they proliferate,at least some of their daughter cells remain as HSCs, so the pool ofstem cells does not become depleted. The other daughters of HSCs(myeloid and lymphoid progenitor cells), however, can each commit to anyof the alternative differentiation pathways that lead to the productionof one or more specific types of blood cells, but cannot self-renew.HSCs give rise to common myeloid progenitor cells and common lymphoidprogenitor cells. This disclosure identifies a cell type downstream ofthe common myeloid progenitor cell, termed a myeloid-erythroidprogenitor cell (MEP), which can give rise to red blood cells andmegakaryocytes (which in turn can differentiate into platelets).

1. Myeloid-Erythroid Progenitor Cells

A “myeloid-erythroid progenitor cell” (or MEP) as used herein, is a cellthat gives rise to megakaryocytes and erythrocytes. It is most commonlyderived from a common myeloid progenitor cell. In some embodiments theMEP is characterized by co-expression of glycophorin A (also known asCD235 in humans) protein (e.g., Uniprot #P02724 in humans), a marker ofthe erythroid lineage, and integrin alpha 2b (CD41 in humans) protein(e.g., Uniprot #P08514 in humans), a marker of megakaryocyte lineage(Klimchenko et. al., Blood, 2009, 114(8):1506-17). In some embodimentsthe MEP does not express CD34.

2. Red Blood Cells

Red blood cells, or erythrocytes, are the most common type of blood celland the vertebrate organism's principal means of delivering oxygen (O₂)to the body tissues via the blood flow through the circulatory system.They take up oxygen in the lungs or gills and release it while squeezingthrough the body's capillaries. The cytoplasm of RBCs is rich inhaemoglobin, an iron-containing biomolecule that can bind oxygen and isresponsible for the blood's red color.

In humans, mature red blood cells are oval and flexible biconcave disks.They lack a cell nucleus and most organelles to accommodate maximumspace for haemoglobin. 2.4 million new erythrocytes are produced persecond. The cells develop in the bone marrow and circulate for about100-120 days in the body before their components are recycled bymacrophages. Each circulation takes about 20 seconds. Approximately aquarter of the cells in the human body are red blood cells.

In some embodiments a “red blood cell” is a cell that co-expressesglycophorin A (also known as CD235 in humans) protein (e.g., Uniprot#P02724 in humans) and transferrin receptor (CD71 in humans) protein(e.g., Uniprot #P02786 in humans) (Hattangadi et. al., Blood, 2011,118(24):6258-68.). In some embodiments the red blood cell furtherexpresses at least one hemoglobin gene. In some embodiments the redblood cell expresses fetal hemoglobin (HbF), and both the alpha and betasubunits of adult type hemoglobin (HbA and HbB). Typically, the cellsresemble hematopoietic progenitor cells, and with maturity, reduce insize and display chromatin condensation (both also signs of maturingRBCs).

3. Megakaryocytes

The megakaryocyte is a bone marrow cell responsible for the productionof blood thrombocytes (platelets), which are necessary for normal bloodclotting. Megakaryocytes normally account for 1 out of 10,000 bonemarrow cells but can increase in number nearly 10-fold during the courseof certain diseases. In general, megakaryocytes are 10 to 15 timeslarger than a typical red blood cell, averaging 50-100 μm in diameter.During its maturation, the megakaryocyte grows in size and replicatesits DNA without cytokinesis in a process called endomitosis. As aresult, the nucleus of the megakaryocyte can become very large andlobulated, which, under a light microscope, can give the falseimpression that there are several nuclei. In some cases, the nucleus maycontain up to 64N DNA, or 32 copies of the normal complement of DNA in ahuman cell. The cytoplasm, just as the platelets that bud off from it,contains α-granula and Dense bodies.

In some embodiments a “megakaryocyte” is a cell that co-expressesintegrin alpha 2b (CD41 in humans) protein (e.g., Uniprot #P08514 inhumans) and glycoprotein Ib (CD42 in humans) protein (e.g., Uniprot#P07359 in humans) (Yu and Cantor, Methods Mol Biol, 2012; 788:291-303).In some embodiments the megakaryocyte expresses GP1balpha. In someembodiments the megakaryocyte further expresses the vWF (vonWillebrand's receptor) as well as CD62P (p selectin). In someembodiments a “megakaryocyte” is a cell that exhibits at least one of acharacteristic polyploidy, the ability to endoreplicate in theproduction of cells up to at least 8N or at least 16N, and noticeableproplatelet extrusions at the surface of the cells (Kaushansky, J ClinInvest, 2005 December; 115(12):3339-47). In some embodiments amegakaryocyte is 2N or 4N.

4. Platelets

Platelets, or thrombocytes, are small, irregularly shaped clear cellfragments (i.e. cells that do not have a nucleus containing DNA), 2-3 μmin diameter, which are derived from fragmentation of precursormegakaryocytes. The average lifespan of a platelet is normally just 5 to9 days. Platelets are a natural source of growth factors. They circulatein the blood of mammals and are involved in hemostasis, leading to theformation of blood clots.

In some embodiments platelets are identified by expression of maturemegakaryocyte markers, such as CD62P. For functionality, plateletadhesion assays are performed or inside out and outside in signalingassays in which the GPIIb/GPIIIa (CD41a/CD42b) complex is interrogated.

D. Methods of Making Megakaryocyte-Erythroid Progenitor Cells (MEPs)

This disclosure also provides methods of making MEPs from stem cells,such as pluripotent stem cells. As demonstrated in the examples, theinventors have established methods and protocols for differentiating apluripotent stem cell into a MEP in culture. Without wishing to be boundby any theory it is believed that the methods demonstrated in theexamples using pluripotent stem cells as the starting cells are broadlyapplicable to the use of hemangioblastic cells or hematopoietic stemcells as well.

In some non-limiting embodients the methods enable at least one ofproducing MEPs at a faster rate and producing MEPs over a longer periodof time in the culture, compared to prior art methods. In someembodiments the pluripotent stem cell is differentiated into a MEP inthe presence of an aryl hydrocarbon receptor (AhR) agonist. Asdemonstrated in the examples, the presence of an AhR agonist in theculture during the differentiation process enables, in some embodiments,exponential production of MEPs.

Differentiation of a stem cell such as a pluripotent stem cell into aMEP is a multi-step process insofar as the pluripotent stem cellundergoes a series of cell fate determinations as it differentiates froma starting pluripotent state, to a hemangioblastic(hematopoietic-endothelial) state, to a multipotent HSC state, to a MEPfate. Of course, if a hemangioblastic (hematopoietic-endothelial) cellor a multipotent HSC cell is used as the starting cell to differentiatea MEP the initial steps of the procedure may be eliminated or modified.By “differentiating a stem cell such as a pluripotent stem cell into aMEP in culture in the presence of an aryl hydrocarbon receptor (AhR)agonist,” is meant that the AhR agonist is present in culture media forat least a sub-period of the total cell culture period. In someembodiments the sub-period is selected from 1 to 12 hours, from 3 to 12hours, from 6 to 12 hours, from 6 to 24 hours, from 12 to 24 hours, from1 to 2 days, from 2 to 4 days, from 3 to 6 days, 1 to 2 weeks, from 2-4weeks, and from 4-8 weeks. In some embodiments the sub-period isselected from at least 1 hour, at least 2 hours, at least 3 hours, atleast 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, atleast 8 hours, at least 9 hours, at least 10 hours, at least 11 hours,at least 12 hours, at least 18 hours, at least 24 hours, at least 36hours, at least 2 days, at least 2 days, at least 3 days, at least 4days, at least 5 days, at least 6 days, at least 1 week, at least 2weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6weeks, at least 7 weeks, and at least 8 weeks.

In some embodiments the AhR agonist is present for the entire cultureperiod. In some embodiments culture of the pluripotent stem cell isinitiated in the absence of the AhR agonist and the AhR agonist is addedafter a period of time selected from at least 1 day, at least 2 days, atleast 3 days, at least 4 days, at least 5 days, at least 6 days, atleast 1 week, and at least 2 weeks. In some embodiments culture of thepluripotent stem cell is initiated in the absence of the AhR agonist andthe AhR agonist is added after a period of time selected from: from 12to 24 hours, from 1 to 2 days, from 1 to 3 days, from 2 to 4 days, from3 to 6 days, from 4 to 7 days, from 5 to 10 days, from 6 to 10 days, andfrom 7 to 10 days.

In some embodiments a pluripotent stem cell is differentiated into a MEPin culture in the presence of at least one protein selected from BMP-4(e.g., Uniprot #P12644 in humans), vVEGF (e.g., Uniprot #P15692 inhumans), WNT3a (e.g., Uniprot #P56704 in humans), bFGF (e.g., Uniprot#P09038 in humans), hSCF (e.g., Uniprot #P21583 in humans), FLT3 (e.g.,Uniprot #P36888 in humans), TPO (e.g., Uniprot #P40225 in humans), andEPOgen (e.g., Uniprot #P01588 in humans). In some embodiments one ormore of those proteins is replaced with another protein having a similaractivity. In some embodiments one of those listed proteins is replacedwith a protein that has at least one characteristic selected from beinga fragment of the listed protein, a fusion protein comprising a fragmentof the listed protein or the whole listed protein, a homologue of thelisted protein, a modified derivative of the listed protein, or a muteinof the listed protein. In some embodiments an AhR agonist is present inthe culture together with the at least one factor. In some embodiments apluripotent stem cell is differentiated into a MEP in culture by amethod that does not comprise culturing in the presence of a AhRagonist.

In some embodiments a pluripotent stem cell is differentiated into a MEPin culture by a method comprising culturing in at least one culturemedia comprising a combination of factors, the culture media and factorcombination comprising a composition selected from:

a) RPMI media supplemented with BMP-4 (e.g., Uniprot #P12644 in humans),VEGF (e.g., Uniprot #P15692 in humans), Wnt3a (e.g., Uniprot #P56704 inhumans), and knockout serum replacement (KOSR) (in some embodiments theratio of BMP-4:VEGF:Wnt3a is about 1:10:5);

b) RPMI media supplemented with BMP-4 (e.g., Uniprot #P12644 in humans),VEGF (e.g., Uniprot #P15692 in humans), bFGF (e.g., Uniprot #P09038 inhumans) and KOSR; (in some embodiments the ratio of BMP-4:VEGF:bFGF isabout 1:10:4);

c) StemPro 34 media supplemented with BMP-4 (e.g., Uniprot #P12644 inhumans), VEGF (e.g., Uniprot #P15692 in humans), and bFGF (e.g., Uniprot#P09038 in humans) (in some embodiments the ratio of BMP-4:VEGF:bFGF isabout 1:10:4);

d) StemPro 34 media supplemented with VEGF (e.g., Uniprot #P15692 inhumans), and bFGF (e.g., Uniprot #P09038 in humans) (in some embodimentsthe ratio of VEGF:bFGF is about 3:1);

e) a mixture of IMDM and Hams F12 media supplemented with B27,N2-supplement, BSA, VEGF (e.g., Uniprot #P15692 in humans), bFGF (e.g.,Uniprot #P09038 in humans), hSCF (e.g., Uniprot #P21583 in humans), andFlt3 ligand (e.g., Uniprot #P36888 in humans) (in some embodiments theratio of VEGF:bFGF:hSCF:Flt3 ligand is about 2:4:4:1);

f) a mixture of IMDM and Hams F12 media supplemented with B27,N2-supplement, BSA, VEGF (e.g., Uniprot #P15692 in humans), bFGF (e.g.,Uniprot #P09038 in humans), hSCF (e.g., Uniprot #P21583 in humans), Flt3ligand (e.g., Uniprot #P36888 in humans), and (e.g., Uniprot #P40225 inhumans), IL-6 (e.g., Uniprot #P05231 in humans), EPOgen (e.g., Uniprot#P01588 in humans) (in some embodiments the ratio of VEGF:bFGF:hSCF:Flt3Ligand:hTPO:IL-6 is about 5:10:10:2.5:10:1); and

g) a mixture of IMDM and Hams F12 media supplemented with B27,N2-supplement, BSA, VEGF (e.g., Uniprot #P15692 in humans), bFGF (e.g.,Uniprot #P09038 in humans), hSCF (e.g., Uniprot #P21583 in humans), Flt3ligand (e.g., Uniprot #P36888 in humans), and hTPO (e.g., Uniprot#P40225 in humans), IL-6 (e.g., Uniprot #P05231 in humans), EPOgen(e.g., Uniprot #P01588 in humans) and an AhR agonist (in someembodiments the ratio of VEGF:bFGF:hSCF:Flt3 Ligand:hTPO:IL-6 is about5:10:10:2.5:10:1).

In some embodiments a pluripotent stem cell is differentiated into a MEPin culture by a method comprising culturing in at least one culturemedia comprising a combination of factors, the culture media and factorcombination comprising a composition selected from:

a) RPMI media supplemented with 4-6 ng/ml BMP-4 (e.g., Uniprot #P12644in humans), 40-60 ng/ml VEGF (e.g., Uniprot #P15692 in humans), 20-30ng/ml Wnt3a (e.g., Uniprot #P56704 in humans), and 10% knockout serumreplacement (KOSR) (in some embodiments the ratio of BMP-4:VEGF:Wnt3a isabout 1:10:5;

b) RPMI media supplemented with 4-6 ng/ml BMP-4 (e.g., Uniprot #P12644in humans), 40-60 ng/ml VEGF (e.g., Uniprot #P15692 in humans), 16-24ng/ml bFGF (e.g., Uniprot #P09038 in humans) and 10% KOSR (in someembodiments the ratio of BMP-4:VEGF:bFGF is about 1:10:4);

c) StemPro 34 media supplemented with 4-6 ng/ml BMP-4 (e.g., Uniprot#P12644 in humans), 40-60 ng/ml VEGF (e.g., Uniprot #P15692 in humans),and 16-24 ng/ml bFGF (e.g., Uniprot #P09038 in humans) (in someembodiments the ratio of BMP-4:VEGF:bFGF is about 1:10:4);

d) StemPro 34 media supplemented with 40-60 ng/ml VEGF (e.g., Uniprot#P15692 in humans), and 4-6 ng/ml bFGF (e.g., Uniprot #P09038 inhumans)(in some embodiments the ratio of VEGF:bFGF is about 3:1);

e) a mixture of IMDM and Hams F12 media supplemented with 1% B27, 0.5%N2-supplement, 0.5% BSA, 12-18 ng/ml VEGF (e.g., Uniprot #P15692 inhumans), 4-6 ng/ml bFGF (e.g., Uniprot #P09038 in humans), 80-120 ng/mlhSCF (e.g., Uniprot #P21583 in humans), and 20-30 ng/ml Flt3 ligand(e.g., Uniprot #P36888 in humans) (in some embodiments the ratio ofVEGF:bFGF:hSCF:Flt3 ligand is about 2:4:4:1);

f) a mixture of IMDM and Hams F12 media supplemented with B27,N2-supplement, BSA, 40-60 ng/ml VEGF (e.g., Uniprot #P15692 in humans),80-120 ng/ml bFGF (e.g., Uniprot #P09038 in humans), 80-120 ng/ml hSCF(e.g., Uniprot #P21583 in humans), 20-30 ng/ml Flt3 ligand (e.g.,Uniprot #P36888 in humans), and 40-60 ng/ml hTPO (e.g., Uniprot #P40225in humans), 8-12 ng/ml IL-6 (e.g., Uniprot #P05231 in humans), 0.5-2U/ml EPOgen (e.g., Uniprot #P01588 in humans) (in some embodiments theratio of VEGF:bFGF:hSCF:Flt3 Ligand:hTPO:IL-6 is about5:10:10:2.5:10:1);

g) a mixture of IMDM and Hams F12 media supplemented with B27,N2-supplement, BSA, 40-60 ng/ml VEGF (e.g., Uniprot #P15692 in humans),80-120 ng/ml bFGF (e.g., Uniprot #P09038 in humans), 80-120 ng/ml hSCF(e.g., Uniprot #P21583 in humans), 20-30 ng/ml Flt3 ligand (e.g.,Uniprot #P36888 in humans), and 40-60 ng/ml hTPO (e.g., Uniprot #P40225in humans), 8-12 ng/ml IL-6 (e.g., Uniprot #P05231 in humans), 0.5-2U/ml EPOgen (e.g., Uniprot #P01588 in humans) (in some embodiments theratio of VEGF:bFGF:hSCF:Flt3 Ligand:hTPO:IL-6 is about 5:10:10:2.5:10:1)and an AhR agonist.

In some embodiments a pluripotent stem cell is differentiated into a MEPin culture by a method comprising culturing in at least one culturemedia comprising a combination of factors, the culture media and factorcombination comprising a composition selected from:

a) RPMI media supplemented with about 5 ng/ml BMP-4 (e.g., Uniprot#P12644 in humans), about 50 ng/ml VEGF (e.g., Uniprot #P15692 inhumans), about 25 ng/ml Wnt3a (e.g., Uniprot #P56704 in humans), andabout 10% knockout serum replacement (KOSR);

b) RPMI media supplemented with about 5 ng/ml BMP-4 (e.g., Uniprot#P12644 in humans), about 50 ng/ml VEGF (e.g., Uniprot #P15692 inhumans), about 20 ng/ml bFGF (e.g., Uniprot #P09038 in humans) and about10% KOSR;

c) StemPro 34 media supplemented with about 5 ng/ml BMP-4 (e.g., Uniprot#P12644 in humans), about 50 ng/ml VEGF (e.g., Uniprot #P15692 inhumans), and about 20 ng/ml bFGF (e.g., Uniprot #P09038 in humans);

d) StemPro 34 media supplemented with about 50 ng/ml VEGF (e.g., Uniprot#P15692 in humans), and about 5 ng/ml bFGF (e.g., Uniprot #P09038 inhumans);

e) a mixture of IMDM and Hams F12 media supplemented with about 1% B27,about 0.5% N2-supplement, about 0.5% BSA, about 15 ng/ml VEGF (e.g.,Uniprot #P15692 in humans), about 5 ng/ml bFGF (e.g., Uniprot #P09038 inhumans), about 100 ng/ml hSCF (e.g., Uniprot #P21583 in humans), andabout 25 ng/ml Flt3 ligand (e.g., Uniprot #P36888 in humans);

f) a mixture of IMDM and Hams F12 media supplemented with about 1% B27,about 0.5% N2-supplement, about 0.5% BSA, about 50 ng/ml VEGF (e.g.,Uniprot #P15692 in humans), about 100 ng/ml bFGF (e.g., Uniprot #P09038in humans), about 100 ng/ml hSCF (e.g., Uniprot #P21583 in humans),about 25 ng/ml Flt3 ligand (e.g., Uniprot #P36888 in humans), and about50 ng/ml hTPO (e.g., Uniprot #P40225 in humans), about 10 ng/ml IL-6(e.g., Uniprot #P05231 in humans), about 0.5 U/ml EPOgen (e.g., Uniprot#P01588 in humans); and

g) a mixture of IMDM and Hams F12 media supplemented with about 1% B27,about 0.5% N2-supplement, about 0.5% BSA, about 50 ng/ml VEGF (e.g.,Uniprot #P15692 in humans), about 100 ng/ml bFGF (e.g., Uniprot #P09038in humans), about 100 ng/ml hSCF (e.g., Uniprot #P21583 in humans),about 25 ng/ml Flt3 ligand (e.g., Uniprot #P36888 in humans), and about50 ng/ml hTPO (e.g., Uniprot #P40225 in humans), about 10 ng/ml IL-6(e.g., Uniprot #P05231 in humans), about 0.5 U/ml EPOgen (e.g., Uniprot#P01588 in humans) and an AhR agonist.

In some embodiments a pluripotent stem cell is differentiated into a MEPin culture by a method comprising:

a) culturing the pluripotent stem cell in RPMI media supplemented withBMP-4, VEGF (e.g., Uniprot #P15692 in humans), Wnt3a (e.g., Uniprot#P56704 in humans), and knockout serum replacement (KOSR);

b) culturing the cell obtained from step a) in RPMI media supplementedwith BMP-4 (e.g., Uniprot #P12644 in humans), VEGF (e.g., Uniprot#P15692 in humans), bFGF (e.g., Uniprot #P09038 in humans) and KOSR;

c) culturing the cell obtained from step b) in StemPro 34 mediasupplemented with BMP-4 (e.g., Uniprot #P12644 in humans), VEGF (e.g.,Uniprot #P15692 in humans), and bFGF (e.g., Uniprot #P09038 in humans);

d) culturing the cell obtained from step c) in StemPro 34 mediasupplemented with VEGF (e.g., Uniprot #P15692 in humans), and bFGF(e.g., Uniprot #P09038 in humans);

e) culturing the cell obtained from step d) in a mixture of IMDM andHams F12 supplemented with B27, N2-supplement, BSA, VEGF (e.g., Uniprot#P15692 in humans), bFGF (e.g., Uniprot #P09038 in humans), hSCF (e.g.,Uniprot #P21583 in humans), and Flt3 ligand (e.g., Uniprot #P36888 inhumans); and

f) culturing the cell obtained from step e) in a mixture of IMDM andHams F12 supplemented with B27, N2-supplement, BSA, VEGF (e.g., Uniprot#P15692 in humans), bFGF (e.g., Uniprot #P09038 in humans), hSCF (e.g.,Uniprot #P21583 in humans), Flt3 ligand (e.g., Uniprot #P36888 inhumans), and hTPO (e.g., Uniprot #P40225 in humans), IL-6 (e.g., Uniprot#P05231 in humans), EPOgen (e.g., Uniprot #P01588 in humans).

In some embodiments the culture media used in step f) further comprisesan AhR agonist.

In some embodiments a pluripotent stem cell is differentiated into a MEPin culture by a method comprising:

a) culturing the pluripotent stem cell in RPMI media supplemented with4-6 ng/ml BMP-4, 40-60 ng/ml VEGF (e.g., Uniprot #P15692 in humans),20-30 ng/ml Wnt3a (e.g., Uniprot #P56704 in humans), and 10% knockoutserum replacement (KOSR)) (in some embodiments the ratio ofBMP-4:VEGF:Wnt3a is about 1:10:5);

b) culturing the cell obtained from step a) in RPMI media supplementedwith 4-6 ng/ml BMP-4 (e.g., Uniprot #P12644 in humans), 40-60 ng/ml VEGF(e.g., Uniprot #P15692 in humans), 16-24 ng/ml bFGF (e.g., Uniprot#P09038 in humans) and 10% KOSR (in some embodiments the ratio ofBMP-4:VEGF:bFGF is about 1:10:4);

c) culturing the cell obtained from step b) in StemPro 34 mediasupplemented with 4-6 ng/ml BMP-4 (e.g., Uniprot #P12644 in humans),40-60 ng/ml VEGF (e.g., Uniprot #P15692 in humans), and 16-24 ng/ml bFGF(e.g., Uniprot #P09038 in humans) (in some embodiments the ratio ofBMP-4:VEGF:bFGF is about 1:10:4);

d) culturing the cell obtained from step c) in StemPro 34 mediasupplemented with 12-18 ng/ml VEGF (e.g., Uniprot #P15692 in humans),and 4-6 ng/ml bFGF (e.g., Uniprot #P09038 in humans) (in someembodiments the ratio of VEGF:bFGF is about 3:1);

e) culturing the cell obtained from step d) in a mixture of IMDM andHams F12 supplemented with 1% B27, 0.5% N2-supplement, 0.5% BSA, 40-60ng/ml VEGF (e.g., Uniprot #P15692 in humans), 80-120 ng/ml bFGF (e.g.,Uniprot #P09038 in humans), 80-120 ng/ml hSCF (e.g., Uniprot #P21583 inhumans), and 20-30 ng/ml Flt3 ligand (e.g., Uniprot #P36888 in humans)(in some embodiments the ratio of VEGF:bFGF:hSCF:Flt3 ligand is about2:4:4:1); and

f) culturing the cell obtained from step e) in a mixture of IMDM andHams F12 supplemented with 1% B27, 0.5% N2-supplement, 0.5% BSA, 40-60ng/ml VEGF (e.g., Uniprot #P15692 in humans), 80-120 ng/ml bFGF (e.g.,Uniprot #P09038 in humans), 80-120 ng/ml hSCF (e.g., Uniprot #P21583 inhumans), 20-30 ng/ml Flt3 ligand (e.g., Uniprot #P36888 in humans), and40-60 ng/ml hTPO (e.g., Uniprot #P40225 in humans), 8-12 ng/ml IL-6(e.g., Uniprot #P05231 in humans), 0.5-2 U/ml EPOgen (e.g., Uniprot#P01588 in humans) (in some embodiments the ratio of VEGF:bFGF:hSCF:Flt3Ligand:hTPO:IL-6 is about 5:10:10:2.5:10:1).

In some embodiments the culture media used in step f) further comprisesan AhR agonist.

In some embodiments a pluripotent stem cell is differentiated into a MEPin culture by a method comprising:

a) culturing the pluripotent stem cell in RPMI media supplemented with 5ng/ml BMP-4, 50 ng/ml VEGF (e.g., Uniprot #P15692 in humans), 25 ng/mlWnt3a (e.g., Uniprot #P56704 in humans), and 10% knockout serumreplacement (KOSR);

b) culturing the cell obtained from step a) in RPMI media supplementedwith 5 ng/ml BMP-4 (e.g., Uniprot #P12644 in humans), 50 ng/ml VEGF(e.g., Uniprot #P15692 in humans), 20 ng/ml bFGF (e.g., Uniprot #P09038in humans) and 10% KOSR;

c) culturing the cell obtained from step b) in StemPro 34 mediasupplemented with 5 ng/ml BMP-4 (e.g., Uniprot #P12644 in humans), 50ng/ml VEGF (e.g., Uniprot #P15692 in humans), and 20 ng/ml bFGF (e.g.,Uniprot #P09038 in humans);

d) culturing the cell obtained from step c) in StemPro 34 mediasupplemented with 15 ng/ml VEGF (e.g., Uniprot #P15692 in humans), and 5ng/ml bFGF (e.g., Uniprot #P09038 in humans);

e) culturing the cell obtained from step d) in a mixture of IMDM andHams F12 supplemented with 1% B27, 0.5% N2-supplement, 0.5% BSA, 50ng/ml VEGF (e.g., Uniprot #P15692 in humans), 100 ng/ml bFGF (e.g.,Uniprot #P09038 in humans), 100 ng/ml hSCF (e.g., Uniprot #P21583 inhumans), and 25 ng/ml Flt3 ligand (e.g., Uniprot #P36888 in humans); and

f) culturing the cell obtained from step e) in a mixture of IMDM andHams F12 supplemented with 1% B27, 0.5% N2-supplement, 0.5% BSA, 50ng/ml VEGF (e.g., Uniprot #P15692 in humans), 100 ng/ml bFGF (e.g.,Uniprot #P09038 in humans), 100 ng/ml hSCF (e.g., Uniprot #P21583 inhumans), 25 ng/ml Flt3 ligand (e.g., Uniprot #P36888 in humans), and 50ng/ml hTPO (e.g., Uniprot #P40225 in humans), 10 ng/ml IL-6 (e.g.,Uniprot #P05231 in humans), and 0.5 U/ml EPOgen (e.g., Uniprot #P01588in humans).

In some embodiments the culture media used in step f) further comprisesan AhR agonist.

In some embodiments, prior to step a), pluripotent stem cells arecultured in iPSC media conditioned on MEFs for 24 hours and supplementedwith Rho Kinase Inhibitor and bFGF.

In some embodiments MEPs made by these or other methods disclosed hereinare isolated.

In some embodiments step a) is for a period of about 2 days, step b) isfor a period of about 1 day, step c) is for a period of about 1 day,step d) is for a period of about 2 days, step e) is for a period ofabout 1 day, and step f) is for a period of about 1 day. In someembodiments the cells are further cultured in the media of step f) for aperiod of: from 1 to 2 days, from 2 to 4 days, from 3 to 6 days, 1 to 2weeks, from 2-4 weeks, and from 4-8 weeks. In some embodiments the cellsare further cultured in the media of step f) for a period of: at least24 hours, at least 36 hours, at least 2 days, at least 2 days, at least3 days, at least 4 days, at least 5 days, at least 6 days, at least 1week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5weeks, at least 6 weeks, at least 7 weeks, and at least 8 weeks. In someembodiments the cells are further cultured in the media of step f) forat least 3 months, at least 6 months, at least 1 year, or indefinitely.In some embodiments the cell culture of step f) is split and frozenstocks are created. Such stocks may be thawed periodically to provide anindefinite supply of a cell culture that forms differentiated MEP cells.

In some embodiments of the methods of this disclosure a culture ofpluripotent stem cells begins to differentiate MEPs within 7 to 10 days.In some embodiments the culture will continue to produce MEPs for atleast 30 days. If during that process the cultured cells are grown inmedia comprising an AhR agonist

In some embodiments the culture does not comprise serum. In someembodiments the culture does not comprise feeder cells. This feeder-freeaspect of such embodiments provides certain advantages in certainsituations. For example, in some such embodiments it reduces the risk ofcontamination of the resultant MEPs, which reduces the risk ofcontamination of red blood cells or platelets made from such MEPs. Thisreduced risk can be desirable in certain applications of the cells.

In some embodiments the proteins used in the protocols in this section Dare modified derivatives and/or muteins of naturally occurring proteins.In some embodiments the protein used are at least 70%, at least 80%, atleast 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or at least 99.5%, identical to the protein sequenceidentified by the Uniprot or other database ID number identified herein.

E. Methods of Making Red Blood Cells

MEPs, including those produced by the methods of this disclosure, havethe potential to differentiate into red blood cells. Accordingly, thisdisclosure also provides methods of making red blood cells, comprisingproviding a MEP and culturing the MEP under conditions sufficient tomake a red blood cell. In some embodiments the methods comprise making aMEP according to a method of this disclosure and culturing the MEP underconditions sufficient to make a red blood cell. In some embodiments, themethods sufficient to make a RBC comprise culturing the MEP in thepresence of an AhR agonist. In some embodiments the conditionssufficient to make a RBC comprise culturing in erythroid specificationmedia. In still further embodiments, the conditions sufficient to make aRBC comprise culturing in erythroid specification media and in thepresence of an AhR agonist. In some embodiments erythroid specificationmedia comprises EPO. (EPO can be from any suitable source known in theart, such as R&D (Catalog #286-EP) or Amgen (EPOgen) commercially, forexample. Erythropoietin has its primary effect on red blood cellprogenitors and precursors (which are found in the bone marrow inhumans) by promoting their survival through protecting these cells fromapoptosis to promote definitive erythropoiesis. Erythropoietin is theprimary erythropoietic factor that cooperates with various other growthfactors (IL-3, IL-6, Glucocorticoids, SCF) involved in the developmentof erythroid lineage from multipotent progenitors. In some embodimentsof this disclosure erythroid specification media comprises EPO. In someembodiments of this disclosure erythroid specification media comprisesat least one additional factor selected from IL-3, IL-6,Glucocorticoids, and SCF. Because the MEPs produced by the methodsdisclosed herein can be transported and can even be frozen, storedand/or transported, this disclosure also enables the distribution ofMEPs made by a method of the disclosure to a different location and/orto a different user, who can then in turn make red blood cells from theMEPs. Accordingly, this disclosure also provides methods of making redblood cells, comprising providing a MEP differentiated in vitro using amethod of this disclosure, and culturing the MEP under conditionssufficient to make a RBC. In some embodiments, the methods sufficient tomake a RBC comprise culturing the MEP in the presence of an AhR agonist.In some embodiments the conditions sufficient to make a RBC compriseculturing in erythroid specification media. In still furtherembodiments, the conditions sufficient to make a RBC comprise culturingin erythroid specification media and in the presence of an AhR agonist.In some embodiments erythroid specification media comprises EPO.

In some embodiments alternative methods of making red blood cells knownin the art are modified to comprise culturing cells in the presence of aAhR agonist to make red blood cells. One exemplary method is thatdisclosed in Feng Ma et. al, PNAS, Sep. 2, 2008, vol. 105, no. 35,p13087-13092. Ma et. al utilize a murine fetal liver stromal cell(mFLSC) layer to differentiate human embryonic stem cells (hESC) intoterminally mature red blood cells. The mFLSC layer was prepared fromembryonic D15 Black 6 mice, expanded and irradiated. hESC were passagedonto wells containing the mFLSC on gelatin and grown in 3 mL of media(α-MEM, 15% FBS, 1 mM glutamine, 1% nonessential amino acids) which waschanged every 3 days. From this culture non-adherent cells were producedand collected on various days. These cells where analyzed by RT-PCR forerythroid gene expression, by immunofluorescence for hemoglobinexpression, and by colony culture for differentiation potential. Theyfound that their cells began expressing erythroid markers as early asday 4 in co-culture and increased expression over time, final time pointD18. β-globin protein expression was detected in individual cells byimmunofluorescence starting and increasing in number from Day 12-18.Further maturation was seen using colony and suspension culture. Cellsfrom the co-culture were plated in 1.2% methylcellulose containing 30%FBS, 1% deionized fraction V BSA, 0.1 mM 2-mercaptoethanol, α-MEM, and ahuman cytokine mixture (100 ng/mL SCF, 10 ng/mL IL-3, 100 ng/mL IL-6, 10ng/mL TPO, 10 ng/mL G-CSF, and 4 U/mL EPO). After 12-14 days erythroidbursts were harvested and grown in a suspension culture of 15% FBS, 0.1mM 2-mercaptoethanol, α-MEM, and the human cytokine mixture listedabove. These cells were analyzed as above for hemoglobins(immunostaining), enucleation (May-Grunwald-Giemsa staining), oxygendissociation (hemox anaylzer), and glucose-6-phosphate dehydrogenaseactivity. They found that these cells were capable of enucleation andhad increased expression of β-globin protein compared to just theco-culture generated cells. These cells bound to oxygen and had similarglucose-6-phosphate dehydrogenase activity as cord blood red bloodcells. Overall, Ma et. al is able to show that by utilizing a mFLSCco-culture followed by erythroid maturation in colony and suspensionculture enucleated red blood cells expression β-globin can be generatedfrom hESC. In some embodiments the method of Ma et al. is modified tocomprise culturing in the presence of an AhR agonist to produce redblood cells from embryonic stem cells.

Another exemplary method is that disclosed in Giarratana et. al, Blood,Nov. 10, 2011, vol. 118, no. 19, p5071-5079. In this paper Giarratanaet. al uses CD34+ cells obtained from human donors to show thefeasibility of cell expansion and use for transfusions. In order toexpand and cause cellular maturation the authors utilize a 3-stepapproach. CD34+ cells are obtained from leukopheresis following bonemarrow stimulation with GM-CSF and CSF. These cells are cultured in EDM(IMDM, 330 ug/ml holo-transferrin, 10 ug/mL rh insulin, 2 U/mL heparin,5% inactivated human plasma) with various cytokines are differentstages. In stage 1 (days 0-7) EDM is supplemented with hydrocortisone,100 ng/mL SCF, 5 ng/mL IL-3, and 3 U/mL EPO. The cells were thenharvested and resuspened in stage 2 (days 7-11) media EDM plus 100 ng/mLSCF and 3 U/mL EPO. On day 11 the cells were once again harvested andresuspended for stage 3 (days 11-18) with EDM media supplemented with 3U/mL of EPO. This methodology allows for cell expansion and maturationof CD34+ cells into reticulocytes. The authors compare their culturedred blood cells (cRBCs) with cord blood and adult blood red blood cellsfor oxygen binding, glucose-6-phosphate dehydrogenase and pyruvatekinase activity, and ability of cells to deform. Their cRBCs behavedvery similarly to cord blood RBCs.

Upon putting the cRBC in vivo (mouse or human) the cells are capable ofcomplete maturation as determined by loss of CD71 expression,organelles, and surface area. They also saw the cells demonstrate abiconcave shape. cRBCs can be stored for normal time frame (4 weeks)without loss of RBC characteristics. In some embodiments the method ofGiarratana et al. is modified to comprise culturing in the presence ofan AhR agonist to produce red blood cells from CD34+ cells.

The methods disclosed herein also enable making a red blood cell fromany MEP from any source. For example, the MEP can be obtained from bonemarrow of a donor. In some embodiments the MEP is first isolated fromother cells present in the bone marrow by, for example, FACS.Alternatively, the whole bone marrow or a fraction thereof can becultured and MEP cell formation stimulated by culturing in the presenceof an AhR agonist. Accordingly, also provided herein are methods ofmaking a red blood cell comprising culturing a MEP in the presence of anAhR agonist. In some embodiments the methods further comprise culturingthe MEP in erythroid specification media.

In some embodiments RBCs made by these or other methods disclosed hereinare isolated. In some embodiments the RBCs are formulated foradministration to a mammal.

F. Methods of Making Megakaryocytes

MEPs, including those produced by the methods of this disclosure, havethe potential to differentiate into megakaryocytes (Mks). Accordingly,this disclosure also provides methods of making megakaryocytes,comprising making a MEP according to a method of this disclosure, andculturing the MEP under conditions sufficient to make a Mk. In someembodiments the conditions sufficient to make a Mk comprise culturingthe MEP in the presence of an AhR modulator. In some embodiments the AhRmodulator is an AhR antagonist. In some embodiments the conditionssufficient to make a Mk comprise culturing the MEP in megakaryocytespecification media. In still further embodiments, the conditionssufficient to make a Mk comprise culturing in megakaryocytespecification media and in the presence of an AhR modulator. In someembodiments the AhR modulator is an AhR antagonist. In some embodimentsmegakaryocyte specification media comprises TPO (for example, Uniprot#P40225 in humans). Human TPO can be acquired commercially from R&D(Catalog #288-TP) or Genentech (Catalog #G140BT), for example. In someembodiments megakaryocyte specification media further comprises stromalderived factor 1 (SDF1).

In some embodiments alternative methods of making Mks known in the artare modified to comprise culturing cells in the presence of a AhRmodulator to make Mks and/or platelets. In some embodiments the AhRmodulator is an AhR antagonist Previously reported differentiationprotocols for human megakaryopoeisis have isolated pluripotent stemcells or human bone marrow for in vitro expansion. Bone marrow protocolsstart with the collection of mononuclear cells from the femur of humansubjects in defined culture conditions (Schattner, M. et al.Thrombopoietin-stimulated ex vivo expansion of human bone marrowmegakaryocytes. Stem Cells. 14, 207-214. (1996). Following in vitroculture and expansion, these cells are sorted using magnetic cellsorting such that the progenitor pool (CD34+) fraction is isolated andused as source material for megakaryocyte differentiation. These cellsare plated on human or murine irradiated bone marrow stroma that serveas a substrate for adhesion and an aid in megakaryocyte maturation forthe CD34+ population. These cultures are grown in media containing humanserum and various permutations of cytokine cocktails containingthrombopoietin (TPO), stem cell factor (SCF), and Interleukin-3 (IL-3).In the seminal report of this protocol, megakaryocyte populations, asdefined by the protein-level expression of CD41a, were observed as earlyas 12 days post-plating. In some embodiments the method of Schattner etal. is modified to comprise culturing in the presence of an AhRantagonist to produce red blood cells from CD34+ cells.

Protocols for differentiating Mks from human pluripotent stem cells (ESCor iPSC) may also be modified to comprise culturing in the presence ofan AhR modulator. In some embodiments the AhR modulator is an AhRantagonist. For example, recent work has proved efficient in optimizingdifferentiation protocols that use human ES or iPS cells as sourcematerial. (Gaur, M. et al. Megakaryocytes derived from human embryonicstem cells: a genetically tractable system to study megakaryocytopoiesisand integrin function. J Thromb Haemost. 4, 436-442. (2006).) The keydifferences with this approach are the added technical complicationsassociated with proper maintenance and passage of pluripotent cells aswell as the challenge of concocting cytokine cocktails that are amenableto inducing hematopoiesis. Gaur et al. solved these issues by usingsimilar strategies as bone marrow protocols. Namely, ESCs were plated onbone marrow stroma and subjected to a high dose of TPO. After 7 days,large colonies thought to contain hematopoietic progenitors werephysically disrupted in order to isolate the progenitor pool in asingle-cell suspension. The cells were plated on a fresh stromalmonolayer and kept in high TPO media until splitting at day 11, whichinvolved a prolonged exposure to trypsin and collagenase, again, toisolate these cells in suspension as best as possible. The cells wereagain replated and found to express megakaryocyte markers and exhibithigh ploidy as early as day 15. In some embodiments the method of Gauret al. is modified to comprise culturing in the presence of an AhRmodulator to produce red blood cells from human pluripotent stem cells.In some embodiments the AhR modulator is an AhR antagonist.

Because the MEPs produced by the methods disclosed herein can betransported and can even be frozen, stored and/or transported, thisdisclosure also enables the distribution of MEPs made by a method of thedisclosure to a different location and/or to a different user, who canthen in turn make megakaryocytes from the MEPs. Accordingly, thisdisclosure also provides methods of making megakaryocytes, comprisingproviding a MEP differentiated in vitro using a method of thisdisclosure, and culturing the MEP under conditions sufficient to make amegakaryocyte. In some embodiments, the methods sufficient to make amegakaryocyte comprise culturing the MEP in the presence of an AhRmodulator. In some embodiments the AhR modulator is an AhR antagonist.In some embodiments the conditions sufficient to make a megakaryocytescomprise culturing in megakaryocyte specification media. In stillfurther embodiments, the conditions sufficient to make a megakaryocytecomprise culturing in megakaryocyte specification media and in thepresence of an AhR modulator. In some embodiments the AhR modulator isan AhR antagonist. In some embodiments megakaryocyte specification mediacomprises TPO.

The methods disclosed herein also enable making a megakaryocyte from anyMEP from any source. For example, the MEP can be obtained from bonemarrow of a donor. In some embodiments the MEP is first isolated fromother cells present in the bone marrow by, for example FACS.Alternatively, the whole bone marrow or a fraction thereof can becultured and MEP cell formation stimulated by culturing in the presenceof an AhR agonist. Accordingly, also provided herein are methods ofmaking a megakaryocyte comprising culturing a MEP from any source in thepresence of an AhR modulator. In some embodiments the AhR modulator isan AhR antagonist. In some embodiments the methods further compriseculturing the MEP in megakaryocyte specification media.

In some embodiments Mks made by these or other methods disclosed hereinare isolated. In some embodiments the Mks are formulated foradministration to a mammal.

G. Methods of Making Platelets

MEPs, including those produced by the methods of this disclosure, havethe potential to differentiate into megakaryocytes, which in turn willnaturally differentiate in culture to form platelets. Accordingly, thisdisclosure also provides methods of making platelets, comprising makinga MEP according to a method of this disclosure, culturing the MEP underconditions sufficient to make a Mk, and culturing the Mk underconditions sufficient for differentiation of a platelet from the Mk. Insome embodiments the conditions sufficient to make a Mk compriseculturing the MEP in the presence of an AhR AhR modulator. In someembodiments the AhR modulator is an AhR antagonist. In some embodimentsthe conditions sufficient to make a Mk comprise culturing the MEP inmegakaryocyte specification media. In still further embodiments, theconditions sufficient to make a Mk comprise culturing in megakaryocytespecification media and in the presence of an AhR modulator. In someembodiments the AhR modulator is an AhR antagonist. In some embodimentsmegakaryocyte specification media comprises TPO.

Because the MEPs produced by the methods disclosed herein can betransported and can even be frozen, stored and/or transported, thisdisclosure also enables the distribution of MEPs made by a method of thedisclosure to a different location and/or to a different user, who canthen in turn make platelets from the MEPs. Accordingly, this disclosurealso provides methods of making platelets, comprising providing a MEPdifferentiated in vitro using a method of this disclosure, culturing theMEP under conditions sufficient to make a megakaryocyte, and culturingthe Mk under conditions sufficient for differentiation of a plateletfrom the Mk. In some embodiments, the methods sufficient to make amegakaryocyte comprise culturing the MEP in the presence of an AhRmodulator. In some embodiments the AhR modulator is an AhR antagonist.In some embodiments the conditions sufficient to make a megakaryocytescomprise culturing in megakaryocyte specification media. In stillfurther embodiments, the conditions sufficient to make a megakaryocytecomprise culturing in megakaryocyte specification media and in thepresence of an AhR modulator. In some embodiments the AhR modulator isan AhR antagonist. In some embodiments megakaryocyte specification mediacomprises TPO.

The methods disclosed herein also enable making a megakaryocyte from anyMEP from any source and thus also allow making a platelet from any MEPsource. For example, the MEP can be obtained from bone marrow of adonor. In some embodiments the MEP is first isolated from other cellspresent in the bone marrow by, for example FACS. Alternatively, thewhole bone marrow or a fraction thereof can be cultured and MEP cellformation stimulated by culturing in the presence of an AhR agonist.Accordingly, also provided herein are methods of making a plateletcomprising culturing a MEP in the presence of an AhR modulator to make aMk and culturing the Mk under conditions sufficient for differentiationof a platelet. In some embodiments the AhR modulator is an AhRantagonist. In some embodiments the methods further comprise culturingthe MEP in megakaryocyte specification media.

In some embodiments platelets made by these or other methods disclosedherein are isolated. In some embodiments the Mks are formulated foradministration to a mammal.

H. Aryl Hydrocarbon Receptor (AhR) Modulators

The Aryl Hydrocarbon Receptor (“AhR”) is a ligand-activated member ofthe family of basic-helix-loop-helix transcription factors that has beenfound to be activated by numerous structurally diverse synthetic andnaturally occurring compounds, such as poly cyclic aromatichydrocarbons, indoles, and flavonoids. In the absence of bound ligand,the AhR is present in a latent conformation in the cytoplasmiccompartment of the cell associated with two molecules of the molecularchaperone heat shock protein 90, an immunophilin-like protein, XAP2, andthe hsp90 interacting protein p23. Ligand binding initiates a cascade ofevents that includes translocation to the nucleus, release of hsp90, andheterodimerization with ARNT and other transcription factor monomers.The ligand bound AhR-ARNT complex is capable of recognizing consensussequences termed dioxin-response elements (“DRE”s) located in thepromoter region of CYP1A1 and other responsive genes, thereby activatingtranscription. Known examples of AhR-associated proteins include, butare not limited to, hsp90 p23, XAP2, p60, hsp70, p48, Re1B, and estrogenreceptor.

The AhR protein contains several domains critical for function and isclassified as a member of the basic helix-loop-helix/Per-Arnt-Sim(bHLH/PAS) family of transcription factors. The bHLH motif is located inthe N-terminal of the protein. Members of the bHLH superfamily have twofunctionally distinctive and highly conserved domains. The first is thebasic-region (b) which is involved in the binding of the transcriptionfactor to DNA. The second is the helix-loop-helix (HLH) region whichfacilitates protein-protein interactions. Also contained with the AhRare two PAS domains, PAS-A and PAS-B, which are stretches of 200-350amino acids that exhibit a high sequence homology to the protein domainsthat were originally found in the Drosophila genes period (Per) andsingle minded (Sim) and in AhR's dimerization partner, the arylhydrocarbon receptor nuclear translocator (ARNT). The PAS domainssupport specific secondary interactions with other PAS domain containingproteins, as is the case with AhR and ARNT, so that heterozygous andhomozygous protein complexes can form. The ligand binding site of AhR iscontained within the PAS-B domain and contains several conservedresidues critical for ligand binding. Finally, a Q-rich domain islocated in the C-terminal region of the protein and is involved inco-activator recruitment and transactivation.

As used herein, “AhR” or “Aryl Hydrocarbon Receptor” refers to anyprotein commonly understood to be a AhR or Aryl Hydrocarbon Receptorfrom any mammal, as well as variants and modified derivatives thereof.In some embodiments the AhR is the human protein identified by Genbankidentifier NP001612, which is hereby incorporated herein by reference.

During canonical signaling, cytosolic AhR binds to a ligand, such as asuitable small molecule, which facilitates AhR translocation to thenucleus and eventually results in de novo transcription of target genes.The promoters of AhR target genes have the responsive element5′-TNGCGTG-3′, termed “AhR response element” or “AHRE” or “Drug responseelement” or “DRE”, or “Xenobiotic response element” or “XRE”. The genesfor xenobiotic-metabolizing enzymes (e.g., cytochrome P450) arewell-known targets of AhR. Hundreds of other genes also have AHREs.Elucidation of the biochemistry of canonical AhR signaling has revealedseveral parameters that can fine-tune AhR activity. These include ligandcharacteristics, adapter molecules and transcriptional co-activators orco-repressors that regulate the extraordinary cell-specific activity ofAhR.

The AhR modulator may be any substance, including without limitation apeptide, a polypeptide, a protein (such as for example an antibody orantibody fragment), a nucleotide, an oligonucleotide, a polynucleotide,a lipid, a sugar, or a naturally occurring or non-naturally occurringderivative thereof. The AhR modulator, whether it also fits within oneor more of the previously listed classes, may be a small organicmolecule or a complex organic molecule.

Molecules with AhR agonist and antagonist activity are well known in theart and may be used in the methods of this disclosure. (See for exampleDenison, M. S., and S. R. Nagy. 2003, “Activation of the arylhydrocarbon receptor by structurally diverse exogenous and endogenouschemicals,” Annu Rev Pharmacol Toxicol 43:309-334; and Nguyen, L. P.,and C. A. Bradfield, 2008, “The search for endogenous activators of thearyl hydrocarbon receptor,” Chemical research in toxicology 21:102-116).

One type of assay that may be used to characterize the activity of suchmolecules or to identify new molecules is an in vitro cell based assay.In one example, the H1G1 mouse hepatoma line is stably transfected withan AhR-driven, green fluorescent protein reporter. Suspected AhR ligandsare added to H1G1 cultures at titered concentrations for 18-48 hours.GFP fluorescence is then quantified in a Luminometer. AhR antagonistactivity is determined by assaying GFP fluorescence in H1G1 cellsfollowing addition of both the compound of interest and a known AhRligand, e.g., β-napthoflavone (BNF). Examples of such assays aredescribed, for example, in Nagy, S. R., et al., 2002, “Identification ofnovel Ah receptor agonists using a high-throughput green fluorescentprotein-based recombinant cell bioassay,” Biochemistry 41:861-868; andNagy, S. R., et al., 2002, “Development of a green fluorescentprotein-based cell bioassay for the rapid and inexpensive detection andcharacterization of ah receptor agonists,” Toxicol Sci 65:200-210, eachof which is hereby incorporated herein by reference for all purposes.See also Garrison et al., 1996, “Species-specific recombinant cell linesas bioassay systems for the detection of2,3,7,8-tetrachlorodibenzo-p-dioxin-like chemicals,” Fundamental andApplied Toxicology, 30:194-203, which is hereby incorporated herein byreference for all purposes.

An exemplary assay that may be used to characterize AhR agonist activityof a test agent is to provide a cell culture comprising MEPs and thenculture the cell culture in the presence of EPO and the test agent andmeasure production of RBCs in the culture. At least one control culturemay optionally be conducted and/or the results of at least one controlcell culture may be referenced. The optional control culture willtypically be a culture in which a similar cell culture comprising MEPsis cultured in the presence of EPO but not the test agent.Alternatively, or in addition, a control cell culture may be conductedor referenced, in which a similar cell culture comprising MEPs iscultured in the presence of EPO and a known AhR agonist, such as FICZ.By assaying for production of RBCs in the cell culture comprising EPOand the test agent, and optionally comparing production of RBCs in thatculture to production of RBCs in at least one of the control cellcultures it is determined whether the test agent has AhR agonistactivity. The test agent is determined by this assay to have AhR agonistactivity if, for example, production of RBCs by the culture comprisingthe test agent and EPO produces more RBCs than a control culturecomprising EPO but not the test agent.

An exemplary assay that may be used to characterize AhR antagonistactivity of a test agent is to provide a cell culture comprising MEPsand then culture the cell culture in at least one of 1) the cell culturecomprising EPO and the test agent; and 2) the cell culture comprisingEPO, a known AhR agonist, and the test agent. At least one controlculture may optionally be conducted and/or the results of at least onecontrol cell culture may be referenced. The optional control culturewill typically be a culture in which a similar cell culture comprisingMEPs is cultured in the presence of EPO but not the test agent, or thepresence of EPO and the known AhR agonist. By assaying for production ofRBCs in the at least one of 1) the cell culture comprising EPO and thetest agent; and 2) the cell culture comprising EPO, the known AhRagonist, and the test agent; and optionally comparing production of RBCsin the at least one culture to production of RBCs in the at least onecontrol cell culture, it is determined whether the test agent has AhRantagonist activity. The test agent is determined by this assay to haveAhR antagonist activity if, for example, production of RBCs by at leastone of 1) the cell culture comprising EPO and the test agent; and 2) thecell culture comprising EPO, a known AhR agonist, and the test agent,produces fewer RBCs than a control culture comprising EPO but not thetest agent, and/or fewer RBCs than a control culture comprising EPO andthe known AhR agonist but not the test agent.

Another exemplary assay that may be used to characterize AhR antagonistactivity of a test agent is to provide a cell culture comprising MEPsand then culture the cell culture in the presence of TPO and the testagent and measure production of Mks and/or platelets in the culture. Atleast one control culture may optionally be conducted and/or the resultsof at least one control cell culture may be referenced. The optionalcontrol culture will typically be a culture in which a similar cellculture comprising MEPs is cultured in the presence of TPO but not thetest agent. Alternatively, or in addition, the control cell culture willbe a culture in which a similar cell culture comprising MEPs is culturedin the presence of TPO and a known AhR agonist, such as FICZ. Byassaying for production of Mks and/or platelets in the cell culturecomprising TPO and the test agent, and optionally comparing productionof Mks and/or platelets in that culture to production of Mks and/orplatelets in at least one of the control cell cultures it is determinedwhether the test agent has AhR antagonist activity. The test agent isdetermined by this assay to have AhR antagonist activity if, forexample, production of Mks and/or platelets by the culture comprisingthe test agent and TPO produces more Mks and/or platelets than a controlculture comprising TPO but not the test agent, and/or more Mks and/orplatelets than a control culture comprising TPO and the known AhRagonist but not the test agent.

In addition, standard physiological, pharmacological and biochemicalprocedures are available for testing agents to identify those thatpossess biological activities that modulate the activity of the AhR.Such assays include, for example, biochemical assays such as bindingassays, fluorescence polarization assays, FRET based coactivatorrecruitment assays (see generally Glickman et al., J. BiomolecularScreening, 7 No. 1 3-10 (2002)), as well as cell based assays includingthe co-transfection assay, the use of LBD-Gal 4 chimeras,protein-protein interaction assays (see, Lehmann. et al., J. Biol Chem.,272(6) 3137-3140 (1997), and gene expression assays.

High throughput screening systems are commercially available (see, e.g.,Zymark Corp., Hopkinton, Mass.; Air Technical Industries, Mentor, Ohio;Beckman Instruments Inc., Fullerton, Calif.; Precision Systems, Inc.,Natick, Mass.) that enable these assays to be run in a high throughputmode. These systems typically automate entire procedures, includingsample and reagent pipetting, liquid dispensing timed incubations, andfinal readings of the microplate in detector(s) appropriate for theassay. These configurable systems provide high throughput and rapidstart up as well as a high degree of flexibility and customization. Themanufacturers of such systems provide detailed protocols for varioushigh throughput systems. Thus, for example, Zymark Corp. providestechnical bulletins describing screening systems for detecting themodulation of gene transcription, ligand binding, and the like.

Assays that do not require washing or liquid separation steps can beused for high throughput screening systems and include biochemicalassays such as fluorescence polarization assays (see for example,Owicki, J., Biomol Screen 2000 Oct; 5(5):297) scintillation proximityassays (SPA) (see for example, Carpenter et al., Methods Mol Biol 2002;190:31-49) and fluorescence resonance energy transfer energy transfer(FRET) or time resolved FRET based coactivator recruitment assays(Mukherjee et al., J Steroid Biochem Mol Biol 2002 July; 81(3):217-25;(Zhou et al., Mol Endocrinol. 1998 October; 12(10):1594-604). Generallysuch assays can be performed using either the full length receptor, orisolated ligand binding domain (LBD).

If a fluorescently labeled ligand is available, fluorescencepolarization assays provide a way of detecting binding of agents to theAhR by measuring changes in fluorescence polarization that occur as aresult of the displacement of a trace amount of the label ligand by theagent.

The ability of an agent to bind to AhR can also be measured in ahomogeneous assay format by assessing the degree to which the agent cancompete off a radiolabelled ligand with known affinity for the receptorusing a scintillation proximity assay (SPA). In this approach, theradioactivity emitted by a radiolabelled agent generates an opticalsignal when it is brought into close proximity to a scintillant such asa Ysi-copper containing bead, to which the AhR is bound. If theradiolabelled agent is displaced from the AhR the amount of lightemitted from the AhR bound scintillant decreases, and this can bereadily detected using standard microplate liquid scintillation platereaders such as, for example, a Wallac MicroBeta reader.

DNA binding assays can be used to evaluate the ability of an agent tomodulate AhR activity. These assays measure the ability of nuclearreceptor proteins, including AhR, to bind to regulatory elements ofgenes known to be modulated by AhR. In general, the assay involvescombining a DNA sequence which can interact with the AhR, and the AhRprotein under conditions, such that the amount of binding of the AhRprotein in the presence or absence of the agent can be measured. In thepresence of an agonist, AhR binds to the regulatory element. Methodsincluding, but not limited to DNAse footprinting, gel shift assays, andchromatin immunoprecipitation can be used to measure the amount of AhRproteins bound to regulatory elements.

In general, a molecule identified as binding to the AhR using one ofthese binding assays may be identified directly in the assay as anagonist or antagonist of AhR, or may be further evaluated, in acell-based assay for example, to determine whether the binding agent isan agonist or antagonist.

In addition a variety of cell based assay methodologies may besuccessfully used in screening assays to identify and profile thespecificity of agents described herein. These approaches include theco-transfection assay, translocation assays, and gene expression assays.

Three basic variants of the co-transfection assay strategy exist,co-transfection assays using full-length AhR, co-transfection assaysusing chimeric AhRs comprising the ligand binding domain of the AhRfused to a heterologous DNA binding domain, and assays based around theuse of the mammalian two hybrid assay system.

The basic co-transfection assay is based on the co-transfection into thecell of an expression plasmid to express the AhR in the cell with areporter plasmid comprising a reporter gene whose expression is underthe control of DNA sequence that is capable of interacting with thatnuclear receptor. Treatment of the transfected cells with an agonist forthe increases the transcriptional activity of that receptor which isreflected by an increase in expression of the reporter gene, which maybe measured by a variety of standard procedures.

Reporter plasmids may be constructed using standard molecular biologicaltechniques by placing cDNA encoding for the reporter gene downstreamfrom a suitable minimal promoter. For example luciferase reporterplasmids may be constructed by placing cDNA encoding firefly luciferaseimmediately downstream from the herpes virus thymidine kinase promoter(located at nucleotides residues −105 to +51 of the thymidine kinasenucleotide sequence) which is linked in turn to the various responseelements.

Numerous methods of co-transfecting the expression and reporter plasmidsare known to those of skill in the art and may be used for theco-transfection assay to introduce the plasmids into a suitable celltype. Typically such a cell will not endogenously express AhR thatinteract with the response elements used in the reporter plasmid.

Numerous reporter gene systems are known in the art and include, forexample, alkaline phosphatase Berger, J., et al (1988) Gene 66 1-10;Kain, S. R. (1997) Methods. Mol. Biol. 63 49-60), 13-galactosidase (See,U.S. Pat. No. 5,070,012, issued Dec., 3, 1991 to Nolan et al., andBronstein, I., et al., (1989) J. Chemilum. Biolum. 4 99-111),chloramphenicol acetyltransferase (See Gorman et al., Mol Cell Biol.(1982) 2 1044-51), 13-glucuronidase, peroxidase, 13-lactamase (U.S. Pat.Nos. 5,741,657 and 5,955,604), catalytic antibodies, luciferases (U.S.Pat. Nos. 5,221,623; 5,683,888; 5,674,713; 5,650,289; 5,843,746) andnaturally fluorescent proteins (Tsien, R. Y. (1998) Annu Rev. Biochem.67 509-44).

The use of chimeras comprising the ligand binding domain (LBD) of theAhR to a heterologous DNA binding domain (DBD) expands the versatilityof cell based assays by directing activation of the AhR in question todefined DNA binding elements recognized by defined DNA binding domain(see W095/18380). This assay expands the utility of cell basedco-transfection assays in cases where the biological response orscreening window using the native DNA binding domain is notsatisfactory.

In general, the methodology is similar to that used with the basicco-transfection assay, except that a chimeric construct is used in placeof the full-length AhR. As with the full-length AhR, treatment of thetransfected cells with an agonist for the AhR LBD increases thetranscriptional activity of the heterologous DNA binding domain which isreflected by an increase in expression of the reporter gene as describedabove. Typically for such chimeric constructs, the DNA binding domainsfrom defined AhRs, or from yeast or bacterially derived transcriptionalregulators such as members of the GAL 4 and Lex A /UmuD super familiesare used.

A third cell based assay of utility for screening agents is a mammaliantwo-hybrid assay that measures the ability of the nuclear receptor tointeract with a cofactor in the presence of a ligand. (See for example,U.S. Pat. Nos. 5,667,973, 5,283,173 and 5,468,614). The basic approachis to create three plasmid constructs that enable the interaction of theAhR with the interacting protein to be coupled to a transcriptionalreadout within a living cell. The first construct is an expressionplasmid for expressing a fusion protein comprising the interactingprotein, or a portion of that protein containing the interacting domain,fused to a GAL4 DNA binding domain. The second expression plasmidcomprises DNA encoding the AhR fused to a strong transcriptionactivation domain such as VP16, and the third construct comprises thereporter plasmid comprising a reporter gene with a minimal promoter andGAL4 upstream activating sequences.

Once all three plasmids are introduced into a cell, the GAL4 DNA bindingdomain encoded in the first construct allows for specific binding of thefusion protein to GAL4 sites upstream of a minimal promoter. Howeverbecause the GAL4 DNA binding domain typically has no strongtranscriptional activation properties in isolation, expression of thereporter gene occurs only at a low level. In the presence of a ligand,the AhR-VP16 fusion protein can bind to the GAL4-interacting proteinfusion protein bringing the strong transcriptional activator VP16 inclose proximity to the GAL4-binding sites and minimal promoter region ofthe reporter gene. This interaction significantly enhances thetranscription of the reporter gene, which can be measured for variousreporter genes as described above. Transcription of the reporter gene isthus driven by the interaction of the interacting protein and AhR in aligand dependent fashion.

An agent can be tested for the ability to induce nuclear localization ofa nuclear protein receptor, such as AhR. Upon binding of an agonist, AhRtranslocates from the cytoplasm to the nucleus. Microscopic techniquescan be used to visualize and quantitate the amount of AhR located in thenucleus. In some embodiments, this assay utilizes a chimeric AhR fusedto a fluorescent protein. Nuclear AhR can also be quantified by westernblotting using AhR-specific antibody and nuclear protein extracts.

An agent can also be evaluated for its ability to increase or decreasethe expression of genes known to be modulated by the AhR in vivo, usingNorthern-blot, RT PCR, oligonucleotide microarray analysis, or highdensity cDNA sequencing to analyze RNA levels. Western-blot analysis canbe used to measure expression of proteins encoded by AhR target genes.Expression of the CYP1B1 gene is modulated by AhR. Additional genesknown to be regulated by the AhR include CYP1A1, CYP1A2, TIPARP, ALDH1or ALDH3, TGF-b, VAV3, IL-18, PROM1, EREG, c-myc, EGR1, GSTA1, SFXN1,and NQO1.

Any agent which is a candidate for modulation of the AhR may be testedby the methods described above. Generally, though not necessarily,agents are tested at several different concentrations and administeredone or more times to optimize the chances that activation of thereceptor will be detected and recognized if present. Typically assaysare performed in triplicate, for example, and vary within experimentalerror by less than about 15%. Each experiment is typically repeatedabout three or more times with similar results.

In some embodiments, the effects of agents and compositions on AhR geneexpression can be evaluated in animals. After the administration ofagents, various tissues can be harvested to determine the effect ofagents on activities directly or indirectly regulated by AhR.

In some embodiments the AhR modulator is an AhR antagonist. Non-limitingexamples of molecules with AhR antagonist activity that may be used inthe methods of this disclosure include:

α-napthoflavone;

1,4-dihydroxyanthraquinone(quinizarin);

1,5-dihydroxyanthraquinone(anthrarufin);

1,8-dihydroxyanthraquinone(danthron);

galangin;

resveratrol;

2-methyl-2H-pyrazole-3-carboxylicacid(2-methyl-4-o-tolylazo-phenyl)-amide (also known as “CH-223191”);

4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol(also known as “SR1”);

N-[2-(3H-indol-3-yl)ethyl]-9-isopropyl-2-(5-methyl-3-pyridyl)purin-6-amine(also known as “GNF351”);

2-(29-amino-39-methoxyphenyl)-oxanaphthalen-4-one (also known as“PD98059”);

(Z)-3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-indolinone (also knownas “TSU-16”);

2-(29-amino-39-methoxyphenyl)-oxanaphthalen-4-one(also known as“PD98059”); and

N-[2-(3H-indol-3-yl)ethyl]-9-isopropyl-2-(5-methyl-3-pyridyl)purin-6-amine;(also known as “GNF351”).

Additional non-limiting examples of molecules with AhR antagonistactivity that may be used in the methods of this disclosure aredescribed in WO 2012/015914, and include2-{[2-(5-bromo-2-furyl)-4-oxo-4H-chromen-3-yl]oxy}acetamide (also knownas “CB7993113”) and CMLD-2166:

In some embodiments the AhR modulator is an AhR agonist. Non-limitingexamples of molecules with AhR agonist activity that may be used in themethods of this disclosure include:

6-formylindolo[3,2-b]carbzole (FICZ);

polycyclic aromatic compounds;

halogenated aromatic hydrocarbons;

planar polychlorinated biphenyls;

purine derivatives;

tryptophan and its metabolites;

lipoxin A4-related eicosanoids;

indirubin;

bilirubin;

amino flavones;

β-naphtoflavone;

1H-benzimidazole;

5-methoxy-2-[[(4-methoxy-3,5-dimethyl-2-pyridinyl)methyl]sulfinyl]benzimidazole(also known as “omeprazole”);

2-methyl-N-[4-nitro-3-(trifluoromethyl)phenyl]-propanamide (also knownas “flutamide”);

3,3-dindole methane;

1-allyl-7-trifluoromethyl-H-indazol-3-yl]-4-methoxyphenol

4-(3-chloro-phenyl)-pyrimidin-2-yl;

2-[[3-(3,4-dimethoxyphenyl)-1-oxo-2-propenyl]amino]benzoic acid (alsoknown as “Tranilast”);

trans-4-[1-(4-[2-(Dimethylamino)ethoxy]phenyl)-2-phenyl-1-butenylphenol

2-(4-chlorophenyl)-4-oxo-4H-chromen-3-yl ethyl carbonate (also known as“CB7950998”); and

90282-01-B9 (T5838025).

The concentration of the AhR agonists and antagonists used will varydepending on how efficiently the agonist or antagonist agonizes orantagonizes the AhR. It is well within the level of skill in the art todetermine an appropriate concentration of AhR agonist or antagonist touse by applying the teachings of this disclosure.

6-formylindolo[3,2-b]carbazole (FICZ) is typically used on cells inculture at a concentration of from 0.02 to 2.0 μM. In some embodimentsFICZ is used at a concentration of from 0.04 to 1.0 uM. In someembodiments FICZ is used at a concentration of from 0.1 to 0.4 uM. Insome embodiments FICZ is used at a concentration of about 0.02 uM ,about 0.04 uM, about 0.06 uM, about 0.8 uM, about 0.1 uM, about 0.11 uM,about 0.12 uM, about 0.13 uM, about 0.14 uM, about 0.15 uM, about 0.16uM, about 0.17 uM, about 0.18 uM, about 0.19 uM, about 0.2 uM, about0.21 uM, about 0.22 uM, about 0.23 uM, about 0.24 uM, about 0.25 uM,about 0.26 uM, about 0.27 uM, about 0.28 uM, about 0.29 uM, about 0.30uM, about 0.4 uM, about 0.5 uM, about 0.6 uM, about 0.7 uM, about 0.8uM, about 0.9 uM, about 1.0 uM.

CH223191 is typically used on cells in culture at a concentration offrom 0.5 to 50 uM. In some embodiments CH223191 is used at aconcentration of from 1.0 to 25 uM. In some embodiments CH223191 is usedat a concentration of from 2.5 to 10 uM. In some embodiments CH223191 isused at a concentration of about 1 uM. In some embodiments CH223191 isused at a concentration of about 2 uM. In some embodiments CH223191 isused at a concentration of about 3 uM. In some embodiments CH223191 isused at a concentration of about 4 uM. In some embodiments CH223191 isused at a concentration of about 5 uM. In some embodiments CH223191 isused at a concentration of about 6 uM. In some embodiments CH223191 isused at a concentration of about 7 uM. In some embodiments CH223191 isused at a concentration of about 8 uM. In some embodiments CH223191 isused at a concentration of about 9 uM. In some embodiments CH223191 isused at a concentration of about 10 uM.

In some embodiments more than one agonist and/or antagonist is used incombination.

With respect to this disclosure it is contemplated that AhR agonismand/or antagonism may be achieved by any method, including withoutlimitation by using a molecule that binds to or interacts with the AhRprotein itself, or a molecule that increases and/or decreases expressionof AhR protein, as well as a molecule that increases and/or decreasescellular events mediated by AhR signaling. Accordingly, also included asAhR modulators are plasmids, DNA, or RNA fragments which themselves, orby virtue of a gene product they encode, alter AhR expression orfunction on transfection, transduction or otherwise entry into mammaliancells.

I. In Vitro Differentiated Hematopoietic Cells

The methods disclosed herein enable, in certain embodiments, theproduction of cell cultures comprising higher numbers of MEPs and higherproportions of MEPs than prior methods. For example, the inventors haveshown that culturing pluripotent stem cells using the methods of thisdisclosure results in cell cultures comprising at least 500,000 MEPs perml. In some embodiments the cultures comprise at least 750,000 MEPs perml. In some embodiments the cultures comprise at least 1.0×10⁶ MEPs perml. In some embodiments the cultures comprise at least 1.1×10⁶ MEPs perml. In some embodiments the cultures comprise at least 1.2×10⁶ MEPs perml. In some embodiments the cultures comprise at least 1.3×10⁶ MEPs perml. In some embodiments the cultures comprise at least 1.4×10⁶ MEPs perml. In some embodiments the cultures comprise at least 1.5×10⁶ MEPs perml. In some embodiments the cultures comprise at least 1.6×10⁶ MEPs perml. In some embodiments the cultures comprise at least 1.7×10⁶ MEPs perml. In some embodiments the cultures comprise at least 1.8×10⁶ MEPs perml. In some embodiments the cultures comprise at least 1.9×10⁶ MEPs perml. In some embodiments the cultures comprise at least 2.0×10⁶ MEPs perml. In some embodiments such methods do not comprise culturing in thepresence of an AhR agonist.

The methods disclosed herein enable, in certain embodiments thatcomprise culturing in the presence of an AhR agonist, the production ofcell cultures comprising even higher numbers of MEPs and higherproportions of MEPs than prior methods. For example, the inventors haveshown that culturing pluripotent stem cells using the methods of thisdisclosure that comprise culturing in the presence of an AhR agonistresults in cell cultures comprising at least 5×10⁶ MEPs per ml. In someembodiments the cultures comprise at least 7.5×10⁶ MEPs per ml. In someembodiments the cultures comprise at least 1.0×10⁷ MEPs per ml. In someembodiments the cultures comprise at least 1.1×10⁷ MEPs per ml. In someembodiments the cultures comprise at least 1.2×10⁷ MEPs per ml. In someembodiments the cultures comprise at least 1.3×10⁷ MEPs per ml. In someembodiments the cultures comprise at least 1.4×10⁷ MEPs per ml. In someembodiments the cultures comprise at least 1.5×10⁷ MEPs per ml. In someembodiments the cultures comprise at least 1.6×10⁷ MEPs per ml. In someembodiments the cultures comprise at least 1.7×10⁷ MEPs per ml. In someembodiments the cultures comprise at least 1.8×10⁷ MEPs per ml. In someembodiments the cultures comprise at least 1.9×10⁷ MEPs per ml. In someembodiments the cultures comprise at least 2.0×10⁷ MEPs per ml.

The methods in certain embodiments also provide for production in asingle cell culture of at least 1×10⁶ MEPs, at least 5×10⁶ MEPs, atleast 1×10⁷ MEPs, at least 5×10⁷ MEPs, at least 1×10⁸ MEPs, or at least5×10⁸ MEPs.

The methods of this disclosure, in certain embodiments, produce cellcultures comprising MEPs in which a high proportion of the total cellsin the culture are MEPs. MEPs represent less than 0.1% of the entirebone marrow population under normal, steady state conditions. Incontrast, the methods of this disclosure produce cell cultures in whichat least 1%, at least 2%, at least 3%, at least 4%, at least 5%, atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, or at least 50% of the cells inthe culture are MEPs.

In some embodiments the compositions of this disclosure comprise atleast 1 of, at least 2 of, or all 3 of:

A) a concentration of MEPs of at least 500,000 MEPs per ml, at least750,000 MEPs per ml, at least 1.0×10⁶ MEPs per ml, at least 1.1×10⁶ MEPsper ml, at least 1.2×10⁶ MEPs per ml, at least 1.3×10⁶ MEPs per ml, atleast 1.4×10⁶ MEPs per ml, at least 1.5×10⁶ MEPs per ml, at least1.6×10⁶ MEPs per ml, at least 1.7×10⁶ MEPs per ml, at least 1.8×10⁶ MEPsper ml, at least 1.9×10⁶ MEPs per ml, at least 2.0×10⁶ MEPs per ml, atleast 5×10⁶ MEPs per ml, at least 7.5×10⁶ MEPs per ml, at least 1.0×10⁷MEPs per ml, at least 1.1×10⁷ MEPs per ml, at least 1.2×10⁷ MEPs per ml,at least 1.3×10⁷ MEPs per ml, at least 1.4×10⁷ MEPs per ml, at least1.5×10⁷ MEPs per ml, at least 1.6×10⁷ MEPs per ml, at least 1.7×10⁷ MEPsper ml, at least 1.8×10⁷ MEPs per ml, at least 1.9×10⁷ MEPs per ml, orat least 2.0×10⁷ MEPs per ml;

B) a total number of MEPs of at least 1×10⁶ MEPs, at least 5×10⁶ MEPs,at least 1×10⁷ MEPs, at least 5×10⁷ MEPs, at least 1×10⁸ MEPs, or atleast 5×10⁸ MEPs; and

C) a proportion of MEPs to total cells in the culture of at least 1%, atleast 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, or at least 50% of the cells in the culture are MEPs.

In some embodiments the culture further comprises at least one of redblood cells, megakaryocytes, and platelets.

In some embodiments the culture is made by a method that does notcomprise sorting cells based on expression of at least one proteinmarker.

J. Therapeutic Uses of In Vitro Differentiated Hematopoietic Cells

The red blood cells and platelets made by the methods of this disclosureand provided by this disclosure may be used therapeutically to providered blood cells or platelets to a patient in need thereof.

1. Red Blood Cells

A common use of red blood cells is to restore oxygen carrying capacityto the blood of a patient that is suffering from anemia due to trauma orother medical problems. Historically they were transfused as part ofwhole blood, but in modern practice the red blood cells and plasmacomponents are transfused separately. The process of identifying acompatible blood product for transfusion is complicated and givingincompatible RBCs to a patient can be fatal.

Red blood cells for transfusion are often mixed with an anticoagulantand usually a storage solution which provides nutrients and preservesthe functionality of the living cells, which are stored at refrigeratedtemperatures. For traditional red blood cell transfusion the cells areseparated from the fluid portion of the blood either after it iscollected from a donor or during the collection process by apheresis.The product is sometimes modified after collection to meet specificpatient requirements.

The main reason a red blood cell transfusion is carried out is to treatanemia. Anemia is a condition that occurs when the body doesn't haveenough red, oxygen-carrying blood cells. This means that the body'stissues and cells aren't getting enough oxygen.

Broadly speaking, anemia can be caused by impaired red blood cellproduction, increased RBC destruction (hemolytic anemias), blood lossand fluid overload (hypervolemia). Several of these may interplay tocause anemia eventually. Indeed, the most common cause of anemia isblood loss, but this usually does not cause any lasting symptoms unlessa relatively impaired RBC production develops.

Anemia from impaired production of red blood cells can be caused by adisturbance of proliferation and differentiation of stem cells (whichcan be caused by pure red cell aplasia, aplastic anemia, anemia of renalfailure in conjunction with insufficient erythropoietin production, andanemia caused by an endocrine disorder), as disturbance of proliferationand maturation of erythroblasts (which can be caused by perniciousanemia, a form of megaloblastic anemia due to vitamin B12 deficiency,anemia of folic acid deficiency, anemia of prematurity, iron deficiencyanemia, thalassemias, congenital dyserythropoietic anemias, and anemiaof renal failure), and other mechanisms (myelophthisic anemia ormyelophthisis, myelodysplastic syndrome, and anemia of chronicinflammation).

Anemia from increased destruction of red blood cells are generallyclassified as hemolytic anemias. These are generally featuring jaundiceand elevated lactate dehydrogenase levels. Anemia from increaseddestruction of red blood cells can be caused by intrinsic(intracorpuscular) abnormalities (which can be caused by hereditaryspherocytosis, hereditary elliptocytosis, abetalipoproteinemia, pyruvatekinase and hexokinase deficiencies, glucose-6-phosphate dehydrogenasedeficiency and glutathione synthetase deficiency, hemoglobinopathies,sickle cell anemia, hemoglobinopathies causing unstable hemoglobins,paroxysmal nocturnal hemoglobinuria, autoimmune disease, and mechanicaltrauma to red cells, such as following open heart surgery.

Anemia from blood loss can occur following trauma or surgery that causesacute blood loss, gastrointestinal track lesions that cause chronicblood loss, gynecological disturbances that cause chronic blood loss,and menstruation.

Red blood cells of this disclosure, such as for example the red bloodcells made using a method disclosed herein, may be used to provide redblood cells to a patient in need thereof, such as a patient sufferingfrom anemia, such as an anemia type described herein. Generally, the redblood cells will be provided to the patient by transfusion in the formof a transfusion composition (sometimes referred to as “packed redcells”). A “transfusion composition” as used herein is a compositioncomprising red blood cells and another factor or factors which providesnutrients and preserves the functionality of the living cells. In someembodiments the transfusion comprises at least one component selectedfrom an anticoagulant. a buffer, and a nutrient. In some embodiments itis a buffered solution comprising at least one nutrient and at least oneanticoagulant.

In some embodiments the patient is in need of treatment for sickle cellanemia.

In some embodiments the patient is in need of treatment for thalassemia.

In some embodiments the red blood cells are blood type matched to theblood type of the patient.

In some embodiments the red blood cells are red blood cells that aredifferentiated in vitro from MEP cells that are differentiated in vitro.In some embodiments the MEP cells are differentiated in vitro from iPSCsderived from the patient. In some embodiments the MEP cells aredifferentiated in vitro from iPSCs derived from the patient and the redblood cells are also differentiated in vitro from iPSCs derived from thepatient. For example, the iPSCs may be iPSCs derived from a somatic cellof the patient, which are then sued to make MEP cells, which are thenoptionally used to make red blood cells having a high degree of geneticidentity to the genome of the patient. In some embodiments the genome ofthe MEPs is at least 99% genetically identical to the genome of thepatient, is at least 99.1% genetically identical to the genome of thepatient, is at least 99.2% genetically identical to the genome of thepatient, is at least 99.3% genetically identical to the genome of thepatient, is at least 99.4% genetically identical to the genome of thepatient, is at least 99.5% genetically identical to the genome of thepatient, is at least 99.6% genetically identical to the genome of thepatient, is at least 99.7% genetically identical to the genome of thepatient, is at least 99.8% genetically identical to the genome of thepatient, is at least 99.9% genetically identical to the genome of thepatient, is at least 99% genetically identical to the genome of thepatient, is at least 99.95% genetically identical to the genome of thepatient, or is identical to the genome of the patient.

2. Platelets

If the number of platelets in the bloodstream is too low, excessivebleeding can occur. However, if the number of platelets is too high,blood clots can form (thrombosis), which may obstruct blood vessels andresult in such events as a stroke, myocardial infarction, pulmonaryembolism or the blockage of blood vessels to other parts of the body,such as the extremities of the arms or legs. An abnormality or diseaseof the platelets is called a thrombocytopathy, which could be either alow number of platelets (thrombocytopenia), a decrease in function ofplatelets (thrombasthenia), or an increase in the number of platelets(thrombocytosis). There are disorders that reduce the number ofplatelets, such as heparin-induced thrombocytopenia (HIT) or thromboticthrombocytopenic purpura (TTP) that typically cause thromboses, orclots, instead of bleeding.

Platelet transfusions are traditionally given to those undergoingchemotherapy for leukemia, multiple myeloma, those with aplastic anemia,AIDS, hypersplenism, ITP, sepsis, bone marrow transplant, radiationtreatment, organ transplant or surgeries such as cardiopulmonary bypass.

Decreased platelet counts caused by decreased production of plateletscan be caused by at least one of: Vitamin B12, deficiency, folic aciddeficiency, leukemia, myelodysplastic syndrome, decreased production ofthrombopoietin by the liver in liver failure, sepsis, Dengue fever, andhereditary syndromes such as congenital amegakaryocytic thrombocytopenia(CAMT), thrombocytopenia absent radius syndrome, Fanconi anemia,Bernard-Soulier syndrome, May-Hegglin anomaly, Grey platelet syndrome,Alport syndrome, and Wiskott-Aldrich syndrome.

Decreased platelet counts caused by increased destruction of plateletscan be caused by at least one of: idiopathic thrombocytopenic purpura,thrombotic thrombocytopenic purpura, hemolytic-uremic syndrome,disseminated intravascular coagulation, paroxysmal nocturnalhemoglobinuria, antiphospholipid syndrome, systemic lupus erythematosus,post-transfusion purpura, neonatal alloimmune thrombocytopenia, splenicsequestration of platelets due to hypersplenism, Dengue fever, andHIV-associated thrombocytopenia.

Thrombocytopenia can also be induced by medications, including valproicacid, methotrexate, carboplatin, interferon, isotretinoin, panobinostat,montelukast sodium, H2 blockers and proton-pump inhibitors.

Platelets of this disclosure, such as for example the platelets madeusing a method disclosed herein, may be used to provide platelets to apatient in need thereof, such as a patient suffering fromthrombocytopenia. Generally, the platelets will be provided to thepatient by transfusion in the form of a transfusion composition. A“transfusion composition” as used herein is a composition comprisingplatelets and at least one second component selected from ananticoagulant, a buffer, and a nutrient.

In some embodiments the platelets are blood type matched to the bloodtype of the patient.

In some embodiments the platelets are platelets that are differentiatedin vitro from MEP cells that are differentiated in vitro. In someembodiments the MEP cells are differentiated in vitro from iPSCs derivedfrom the patient. In some embodiments the MEP cells are differentiatedin vitro from iPSCs derived from the patient and the platelets are alsodifferentiated in vitro from iPSCs derived from the patient. Forexample, the iPSCs may be iPSCs derived from a somatic cell of thepatient, which are then used to make MEP cells, which are thenoptionally used to make megakaryocytes that in turn differentiate intoplatelets. The megakaryocytes in many such embodiments have a highdegree of genetic identity to the genome of the patient. In someembodiments the genome of the MEPs is at least 99% genetically identicalto the genome of the patient, is at least 99.1% genetically identical tothe genome of the patient, is at least 99.2% genetically identical tothe genome of the patient, is at least 99.3% genetically identical tothe genome of the patient, is at least 99.4% genetically identical tothe genome of the patient, is at least 99.5% genetically identical tothe genome of the patient, is at least 99.6% genetically identical tothe genome of the patient, is at least 99.7% genetically identical tothe genome of the patient, is at least 99.8% genetically identical tothe genome of the patient, is at least 99.9% genetically identical tothe genome of the patient, is at least 99% genetically identical to thegenome of the patient, is at least 99.95% genetically identical to thegenome of the patient, or is identical to the genome of the patient.

K. Analysis of Agents Using In Vitro Differentiated Hematopoietic Cells

The methods of this disclosure allow for production of vast quantitiesof MEPs, red blood cells, megakaryocytes, and platelets. As such, thesemethods and cells made by them have many features that in certainembodiments will make them advantageous for use in screening proceduresthat utilize at least one cell type selected from MEP, red blood cell,megakaryocyte, and platelet. For example, in some embodiments the use ofat least one cell type selected from MEP, red blood cell, megakaryocyte,and platelet differentiated from a known source of pluripotent stemcells provides a high degree of uniformity to an assay involving aplurality of test agents. In some embodiments the at least one cell typeselected from MEP, red blood cell, megakaryocyte, and platelet aredifferentiated from pluripotent stem cells that share at least onecommon genetic factor selected from a blood type genotype, a minorhistocompatibility antigen genotype, and a major histocompatibilitygenotype. In some embodiments the at least one cell type selected fromMEP, red blood cell, megakaryocyte, and platelet are differentiated frompluripotent stem cells comprising genomes that are at least 99%genetically identical to each other, at least 99.1% geneticallyidentical to each other, at least 99.2% genetically identical to eachother, at least 99.3% genetically identical to each other, at least99.4% genetically identical to each other, at least 99.5% geneticallyidentical to each other, at least 99.6% genetically identical to eachother, at least 99.7% genetically identical to each other, at least99.8% genetically identical to each other, at least 99.9% geneticallyidentical to each other, at least 99.95% genetically identical to eachother, or are genetically identical to each other.

Accordingly, provided herein are methods of screening a test agent foran effect on at least one cell type selected from MEP, red blood cell,megakaryocyte, and platelet, the at least one cell type made by a methodof this disclosure or provided by this disclosure. In some embodimentsthe method comprises: a) making the at least one cell type selected fromMEP, red blood cell, megakaryocyte, and platelet by a method of thisdisclosure; b) contacting the at least one cell type selected from MEP,red blood cell, megakaryocyte, and platelet with the test agent; and c)observing a change in the at least one cell type selected from MEP, redblood cell, megakaryocyte, and platelet.

In some embodiments the method further comprises comparing the change inthe at least one cell type selected from MEP, red blood cell,megakaryocyte, and platelet in the presence of the test agent to asimilar cell grown in control conditions that do not comprise the testagent.

In some embodiments the method further comprises d) contacting the atleast one cell type selected from MEP, red blood cell, megakaryocyte,and platelet with a control agent; f) observing a change in the at leastone cell type selected from MEP, red blood cell, megakaryocyte, andplatelet in the presence of the control agent, and g) comparing thechange in the at least one cell type selected from MEP, red blood cell,megakaryocyte, and platelet in the presence of the control agent to thechange in the at least one cell type selected from MEP, red blood cell,megakaryocyte, and platelet in the presence of the test agent.

The change in the at least one cell type selected from MEP, red bloodcell, megakaryocyte, and platelet in the presence of the test agent isin some embodiments selected from a change in rate of cellproliferation, a change in rate of cell death, a change in theexpression level of at least one gene, and a change in the level of atleast one protein in the cell. In some embodiments the change is achange in the level of a marker of toxicity. The change is in someembodiments selected from an increase and a decrease in rate or level.

L. Therapeutic Uses of AhR Modulators

As shown in the Examples, AhR antagonism results in Mk specification andproduction in cultures of MEPs. The Examples also show thatadministration of an effective amount of an AhR agonist to a mammalincreases the platelet count of the mammal. Taken together these datademonstrate that both AhR agonism and antagonism play a role in theprocess: AhR agonism increases the number of MEPs, which can then go onto produce more Mks and more platelets (as well as more RBCs). Moreover,AhR antagonists appear to act on aspecified Mks to increaseendoreplication and platelet production.

Accordingly, this disclosure provides methods of increasing the plateletcount of a mammal. In some embodiments the methods compriseadministering an effective amount of an AhR modulator to the mammal. Insome embodiments the methods comprise administering an effective amountof an AhR agonist to the mammal. In some embodiments the methodscomprise administering an effective amount of an AhR antagonist to themammal. In some embodiments the methods comprise administering aneffective amount of an AhR agonist and an effective amount of an AhRantagonist to the mammal. In some embodiments in which both an AhRagonist and an AhR antagonist are administered, the AhR agonst and AhRantagonist are co-administred. In some embodiments in which both an AhRagonist and an AhR antagonist are administered, the AhR agonst and AhRantagonist are administered separately. Increasing platelet counts inmammals is useful in many ways, including, by way of example, to treat amammal suffering from and/or at risk of thrombocytopenia. Accordingly,this disclosure also provides methods of treating thrombocytopenia in amammal. In some embodiments the methods comprise administering aneffective amount of an AhR agonist to the mammal.

Pharmaceutical compositions for use in the methods of treatment andmethods of increasing the platelet count in a mammal herein areformulated to contain therapeutically effective amounts of at least oneAhR receptor modulator. The pharmaceutical compositions are useful, forexample, in the treatment of at least one disease state characterized bya low platelet count.

In some embodiments, the at least one AhR receptor modulator isformulated into a suitable pharmaceutical preparation such as solutions,suspensions, tablets, dispersible tablets, pills, capsules, powders,sustained release formulations or elixirs, for oral administration or insterile solutions or suspensions for parenteral administration, as wellas transdermal patch preparation and dry powder inhalers. Typically theAhR modulator described above is formulated into pharmaceuticalcompositions using techniques and procedures well known in the art (see,e.g., Ansel Introduction to Pharmaceutical Dosage Forms, Fourth Edition1985, 126).

In the compositions, effective concentrations of one or more AhRmodulators or pharmaceutically acceptable derivatives is (are) mixedwith a suitable pharmaceutical carrier or vehicle.

Pharmaceutically acceptable derivatives include acids, bases, enolethers and esters, salts, esters, hydrates, solvates and prodrug foul's.The derivative is selected such that its pharmacokinetic properties aresuperior with respect to at least one characteristic to thecorresponding neutral agent. The AhR modulator may be derivatized priorto formulation.

The concentrations of the AhR modulator in the compositions areeffective for delivery of an amount, upon administration, that treatsone or more of the symptoms of at least one disease state characterizedby a reduced platelet count and/or a reduction in normal plateletfunction, for example.

Typically, by way of example and without limitation, the compositionsare formulated for single dosage administration. To formulate acomposition, the weight fraction of AhR modulator is dissolved,suspended, dispersed or otherwise mixed in a selected vehicle at aneffective concentration such that the treated condition is relieved orameliorated. Pharmaceutical carriers or vehicles suitable foradministration of the AhR modulator include any such carriers known tothose skilled in the art to be suitable for the particular mode ofadministration.

In addition, the AhR modulator may be formulated as the sole activeagent in the composition or may be combined with other active agents.Liposomal suspensions, including tissue-targeted liposomes, may also besuitable as pharmaceutically acceptable carriers. These may be preparedaccording to methods known to those skilled in the art. For example,liposome formulations may be prepared as described in U.S. Pat. No.4,522,811. Briefly, liposomes such as multilamellar vesicles (MLV's) maybe formed by drying down egg phosphatidyl choline and brain phosphatidylserine (7:3 molar ratio) on the inside of a flask. A solution of a AhRmodulator provided herein in phosphate buffered saline lacking divalentcations (PBS) is added and the flask shaken until the lipid film isdispersed. The resulting vesicles are washed to remove unencapsulatedAhR modulator, pelleted by centrifugation, and then resuspended in PBS.

The active AhR modulator is included in the pharmaceutically acceptablecarrier in an amount sufficient to exert a therapeutically useful effectin the absence of undesirable side effects on the patient treated. Thetherapeutically effective concentration may be determined empirically bytesting the agents in in vitro and in vivo systems described herein andin International Patent Application Publication Nos. 99/27365 and00/25134 and then extrapolated there from for dosages for humans.

The concentration of active AhR modulator in the pharmaceuticalcomposition will depend on absorption, inactivation and excretion ratesof the active agent, the physicochemical characteristics of the agent,the dosage schedule, and amount administered as well as other factorsknown to those of skill in the art. For example, the amount that isdelivered is sufficient to treat at least one disease statecharacterized by at least one of reduced platelet count and reducedplatelet function, as described herein.

Typically a therapeutically effective dosage should produce a serumconcentration of active agent of from about 0.1 ng/ml to about 50-100μg/ml, for example. The pharmaceutical compositions typically shouldprovide a dosage of from about 0.001 mg to about 2000 mg of AhRmodulator per kilogram of body weight per day, such as from about 0.01mg to about 200 mg of AhR modulator per kilogram of body weight per day,or from about 0.1 mg to about 20 mg of AhR modulator per kilogram ofbody weight per day, or from about 1 mg to about 10 mg of AhR modulatorper kilogram of body weight per day, or from about 1 mg to about 5 mg ofAhR modulator per kilogram of body weight per day. Pharmaceutical dosageunit forms are prepared to provide from about 1 mg to about 1000 mg,such as from about 10 to about 500 mg of the active agent or acombination of agents per dosage unit form.

The active agent may be administered at once, or may be divided into anumber of smaller doses to be administered at intervals of time. It isunderstood that the precise dosage and duration of treatment is afunction of the disease state being treated and may be determinedempirically using known testing protocols or by extrapolation from invivo or in vitro test data. It is to be noted that concentrations anddosage values may also vary with the severity of the condition to bealleviated. It is to be further understood that for any particularsubject, specific dosage regimens should be adjusted over time accordingto the individual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat the concentration ranges set forth herein are exemplary only andare not intended to limit the scope or practice of the claimed methods.

Thus, effective concentrations or amounts of one or more AhR Xmodulators or pharmaceutically acceptable derivatives thereof are mixedwith a suitable pharmaceutical carrier or vehicle for systemic, topicalor local administration to form pharmaceutical compositions. AhRmodulators are included in an amount effective for treating at least onedisease state characterized by reduced platelet count and/or plateletfunction. The concentration of active agent in the composition willdepend on absorption, inactivation, excretion rates of the active agent,the dosage schedule, amount administered, particular formulation as wellas other factors known to those of skill in the art.

The compositions are intended to be administered by a suitable route,including by way of example and without limitation orally, parenterally,rectally, topically and locally. For oral administration, capsules andtablets can be used. The compositions are in liquid, semi-liquid orsolid foul and are formulated in a manner suitable for each route ofadministration.

Solutions or suspensions used for parenteral, intradermal, subcutaneous,or topical application can include any of the following components, inany combination: a sterile diluent, including by way of example withoutlimitation, water for injection, saline solution, fixed oil,polyethylene glycol, glycerine, propylene glycol or other syntheticsolvent; antimicrobial agents, such as benzyl alcohol and methylparabens; antioxidants, such as ascorbic acid and sodium bisulfite;chelating agents, such as ethylenediaminetetraacetic acid (EDTA);buffers, such as acetates, citrates and phosphates; and agents for theadjustment of tonicity such as sodium chloride or dextrose. Parenteralpreparations can be enclosed in ampoules, disposable syringes or singleor multiple dose vials made of glass, plastic or other suitablematerial.

In instances in which the agents exhibit insufficient solubility,methods for solubilizing agents may be used. Such methods are known tothose of skill in this art, and include, but are not limited to, usingco-solvents, such as dimethylsulfoxide (DMSO), using surfactants, suchas TWEEN®, or dissolution in aqueous sodium bicarbonate.Pharmaceutically acceptable derivatives of the agents may also be usedin formulating effective pharmaceutical compositions.

Upon mixing or addition of the agent(s), the resulting mixture may be asolution, suspension, emulsion or the like. The form of the resultingmixture depends upon a number of factors, including the intended mode ofadministration and the solubility of the agent in the selected carrieror vehicle. The effective concentration is sufficient for treating oneor more symptoms of at least one disease state characterized by reducedplatelet count and/or function and may be empirically determined.

The pharmaceutical compositions are provided for administration tohumans and animals in unit dosage forms, such as tablets, capsules,pills, powders, granules, sterile parenteral solutions or suspensions,and oral solutions or suspensions, and oil-water emulsions containingsuitable quantities of the agents or pharmaceutically acceptablederivatives thereof. The pharmaceutically therapeutically active agentsand derivatives thereof are typically formulated and administered inunit-dosage forms or multiple-dosage forms. Unit-dose foams as usedherein refers to physically discrete units suitable for human and animalsubjects and packaged individually as is known in the art. Eachunit-dose contains a predetermined quantity of the therapeuticallyactive agent sufficient to produce the desired therapeutic effect, inassociation with the required pharmaceutical carrier, vehicle ordiluent. Examples of unit-dose forms include ampoules and syringes andindividually packaged tablets or capsules. Unit-dose forms may beadministered in fractions or multiples thereof. A multiple-dose form isa plurality of identical unit-dosage forms packaged in a singlecontainer to be administered in segregated unit-dose form. Examples ofmultiple-dose forms include vials, bottles of tablets or capsules orbottles of pints or gallons. Hence, multiple dose form is a multiple ofunit-doses which are not segregated in packaging.

The composition can contain along with the active agent, for example andwithout limitation: a diluent such as lactose, sucrose, dicalciumphosphate, or carboxymethylcellulose; a lubricant, such as magnesiumstearate, calcium stearate and talc; and a binder such as starch,natural gums, such as gum acacia gelatin, glucose, molasses,polyvinylpyrrolidone, celluloses and derivatives thereof, povidone,crospovidones and other such binders known to those of skill in the art.Liquid pharmaceutically administrable compositions can, for example, beprepared by dissolving, dispersing, or otherwise mixing an active agentas defined above and optional pharmaceutical adjuvants in a carrier,such as, by way of example and without limitation, water, saline,aqueous dextrose, glycerol, glycols, ethanol, and the like, to therebyform a solution or suspension. If desired, the pharmaceuticalcomposition to be administered may also contain minor amounts ofnontoxic auxiliary substances such as wetting agents, emulsifyingagents, or solubilizing agents, pH buffering agents and the like, suchas, by way of example and without limitation, acetate, sodium citrate,cyclodextrin derivatives, sorbitan monolaurate, triethanolamine sodiumacetate, triethanolamine oleate, and other such agents. Actual methodsof preparing such dosage forms are known, or will be apparent, to thoseskilled in this art; for example, see Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa., 15th Edition, 1975. Thecomposition or formulation to be administered will, in any event,contain a quantity of the active agent in an amount sufficient toalleviate the symptoms of the treated subject.

Dosage forms or compositions containing active agent in the range of0.005% to 100% with the balance made up from non-toxic carrier may beprepared. For oral administration, a pharmaceutically acceptablenon-toxic composition is formed by the incorporation of any of thenormally employed excipients, such as, for example and withoutlimitation, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, talcum, cellulose derivatives, sodiumcrosscarmellose, glucose, sucrose, magnesium carbonate or sodiumsaccharin. Such compositions include solutions, suspensions, tablets,capsules, powders and sustained release formulations, such as, but notlimited to, implants and microencapsulated delivery systems, andbiodegradable, biocompatible polymers, such as collagen, ethylene vinylacetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylacticacid and others. Methods for preparation of these compositions are knownto those skilled in the art. The contemplated compositions may contain0.001%-100% active agent, such as 0.1-85%, or such as 75-95%.

The active agents or pharmaceutically acceptable derivatives may beprepared with carriers that protect the agent against rapid eliminationfrom the body, such as time release formulations or coatings. Thecompositions may include other active agents to obtain desiredcombinations of properties. AhR modulators or pharmaceuticallyacceptable derivatives thereof, may also be advantageously administeredfor therapeutic or prophylactic purposes together with anotherpharmacological agent known in the general art to be of value intreating at least one disease state characterized by reduced plateletcounts and/or function.

Oral pharmaceutical dosage forms include, by way of example and withoutlimitation, solid, gel and liquid. Solid dosage forms include tablets,capsules, granules, and bulk powders. Oral tablets include compressed,chewable lozenges and tablets which may be enteric-coated, sugar-coatedor film-coated. Capsules may be hard or soft gelatin capsules, whilegranules and powders may be provided in non-effervescent or effervescentforms with the combination of other ingredients known to those skilledin the art.

In some embodiments, the formulations are solid dosage forms, such ascapsules or tablets. The tablets, pills, capsules, troches and the likecan contain any of the following ingredients, or agents of a similarnature: a binder; a diluent; a disintegrating agent; a lubricant; aglidant; a sweetening agent; and a flavoring agent.

Examples of binders include, by way of example and without limitation,microcrystalline cellulose, gum tragacanth, glucose solution, acaciamucilage, gelatin solution, sucrose, and starch paste. Lubricantsinclude, by way of example and without limitation, talc, starch,magnesium or calcium stearate, lycopodium and stearic acid. Diluentsinclude, by way of example and without limitation, lactose, sucrose,starch, kaolin, salt, mannitol, and dicalcium phosphate. Glidantsinclude, by way of example and without limitation, colloidal silicondioxide. Disintegrating agents include, by way of example and withoutlimitation, crosscarmellose sodium, sodium starch glycolate, alginicacid, corn starch, potato starch, bentonite, methylcellulose, agar andcarboxymethylcellulose. Coloring agents include, by way of example andwithout limitation, any of the approved certified water soluble Fl) andC dyes, mixtures thereof; and water insoluble ID and C dyes suspended onalumina hydrate. Sweetening agents include, by way of example andwithout limitation, sucrose, lactose, mannitol and artificial sweeteningagents such as saccharin, and any number of spray dried flavors.Flavoring agents include, by way of example and without limitation,natural flavors extracted from plants such as fruits and syntheticblends of agents which produce a pleasant sensation, such as, but notlimited to peppermint and methyl salicylate. Wetting agents include, byway of example and without limitation, propylene glycol monostearate,sorbitan monooleate, diethylene glycol monolaurate, and polyoxyethylenelaural ether. Emetic-coatings include, by way of example and withoutlimitation, fatty acids, fats, waxes, shellac, ammoniated shellac andcellulose acetate phthalates. Film coatings include, by way of exampleand without limitation, hydroxyethylcellulose, sodiumcarboxymethylcellulose, polyethylene glycol 4000 and cellulose acetatephthalate.

If oral administration is desired, the agent could be provided in acomposition that protects it from the acidic environment of the stomach.For example, the composition can be formulated in an enteric coatingthat maintains its integrity in the stomach and releases the activeagent in the intestine. The composition may also be formulated incombination with an antacid or other such ingredient.

When the dosage unit form is a capsule, it can contain, in addition tomaterial of the above type, a liquid carrier such as a fatty oil. Inaddition, dosage unit forms can contain various other materials whichmodify the physical form of the dosage unit, for example, coatings ofsugar and other enteric agents. The agents can also be administered as acomponent of an elixir, suspension, syrup, wafer, sprinkle, chewing gumor the like. A syrup may contain, in addition to the active agents,sucrose as a sweetening agent and certain preservatives, dyes andcolorings and flavors.

The active materials can also be mixed with other active materials whichdo not impair the desired action, or with materials that supplement thedesired action, such as antacids, H2 blockers, and diuretics.

Pharmaceutically acceptable carriers included in tablets are binders,lubricants, diluents, disintegrating agents, coloring agents, flavoringagents, and wetting agents. Enteric-coated tablets, because of theenteric-coating, resist the action of stomach acid and dissolve ordisintegrate in the neutral or alkaline intestines. Sugar-coated tabletsare compressed tablets to which different layers of pharmaceuticallyacceptable substances are applied. Film-coated tablets are compressedtablets which have been coated with a polymer or other suitable coating.Multiple compressed tablets are compressed tablets made by more than onecompression cycle utilizing the pharmaceutically acceptable substancespreviously mentioned. Coloring agents may also be used in the abovedosage forms. Flavoring and sweetening agents are used in compressedtablets, sugar-coated, multiple compressed and chewable tablets.Flavoring and sweetening agents are useful in the formation of chewabletablets and lozenges.

Liquid oral dosage forms include aqueous solutions, emulsions,suspensions, solutions and/or suspensions reconstituted fromnon-effervescent granules and effervescent preparations reconstitutedfrom effervescent granules. Aqueous solutions include, for example,elixirs and syrups. Emulsions are either oil-in-water or water-in-oil.

Elixirs are clear, sweetened, hydroalcoholic preparations.Pharmaceutically acceptable carriers used in elixirs include solvents.Syrups are concentrated aqueous solutions of a sugar, for example,sucrose, and may contain a preservative. An emulsion is a two-phasesystem in which one liquid is dispersed in the form of small globulesthroughout another liquid. Pharmaceutically acceptable carriers used inemulsions are non-aqueous liquids, emulsifying agents and preservatives.Suspensions use pharmaceutically acceptable suspending agents andpreservatives. Pharmaceutically acceptable substances used innon-effervescent granules, to be reconstituted into a liquid oral dosageform, include diluents, sweeteners and wetting agents. Pharmaceuticallyacceptable substances used in effervescent granules, to be reconstitutedinto a liquid oral dosage form, include organic acids and a source ofcarbon dioxide. Coloring and flavoring agents may be used in any of theabove dosage forms.

Solvents, include by way of example and without limitation, glycerin,sorbitol, ethyl alcohol and syrup. Examples of preservatives includewithout limitation glycerin, methyl and propylparaben, benzoic add,sodium benzoate and alcohol. Non-aqueous liquids utilized in emulsions,include by way of example and without limitation, mineral oil andcottonseed oil. Emulsifying agents, include by way of example andwithout limitation, gelatin, acacia, tragacanth, bentonite, andsurfactants such as polyoxyethylene sorbitan monooleate. Suspendingagents include, by way of example and without limitation, sodiumcarboxymethylcellulose, pectin, tragacanth, Veegum and acacia. Diluentsinclude, by way of example and without limitation, lactose and sucrose.Sweetening agents include, by way of example and without limitation,sucrose, syrups, glycerin and artificial sweetening agents such assaccharin. Wetting agents, include by way of example and withoutlimitation, propylene glycol monostearate, sorbitan monooleate,diethylene glycol monolaurate, and polyoxyethylene lauryl ether. Organicacids include, by way of example and without limitation, citric andtartaric acid. Sources of carbon dioxide include, by way of example andwithout limitation, sodium bicarbonate and sodium carbonate. Coloringagents include, by way of example and without limitation, any of theapproved certified water soluble FD and C dyes, and mixtures thereof.Flavoring agents include, by way of example and without limitation,natural flavors extracted from plants such fruits, and synthetic blendsof agents which produce a pleasant taste sensation.

For a solid dosage form, the solution or suspension, in for examplepropylene carbonate, vegetable oils or triglycerides, is encapsulated ina gelatin capsule. Such solutions, and the preparation and encapsulationthereof, are disclosed in U.S. Pat. Nos. 4,328,245; 4,409,239; and4,410,545. For a liquid dosage form, the solution, for example in apolyethylene glycol, may be diluted with a sufficient quantity of apharmaceutically acceptable liquid carrier, e.g., water, to be easilymeasured for administration.

Alternatively, liquid or semi-solid oral formulations may be prepared bydissolving or dispersing the active agent or salt in vegetable oils,glycols, triglycerides, propylene glycol esters (e.g., propylenecarbonate) and other such carriers, and encapsulating these solutions orsuspensions in hard or soft gelatin capsule shells. Other usefulformulations include those set forth in U.S. Pat. No. Re 28,819 and U.S.Pat. No. 4,358,603. Briefly, such formulations include, but are notlimited to, those containing an agent provided herein, a dialkylatedmono- or poly-alkylene glycol, including, but not limited to,1,2-dimethoxymethane, diglyme, triglyme, tetraglyme, polyethyleneglycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether,polyethylene glycol-750-dimethyl ether wherein 350, 550 and 750 refer tothe approximate average molecular weight of the polyethylene glycol, andone or more antioxidants, such as butylated hydroxytoluene (BHT),butylated hydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone,hydroxycoumarins, ethanolamine, lecithin, cephalin, ascorbic acid, malicacid, sorbitol, phosphoric acid, thiodipropionic acid and its esters,and dithiocarbamates.

Other formulations include, but are not limited to, aqueous alcoholicsolutions including a pharmaceutically acceptable acetal. Alcohols usedin these formulations are any pharmaceutically acceptable water-misciblesolvents having one or more hydroxyl groups, including, but not limitedto, propylene glycol and ethanol. Acetals include, but are not limitedto, di(lower alkyl) acetals of lower alkyl aldehydes such asacetaldehyde diethyl acetal.

Tablets and capsules formulations may be coated as known by those ofskill in the art in order to modify or sustain dissolution of the activeingredient. Thus, for example and without limitation, they may be coatedwith a conventional enterically digestible coating, such asphenylsalicylate, waxes and cellulose acetate phthalate.

Parenteral administration, generally characterized by injection, eithersubcutaneously, intramuscularly or intravenously is also contemplatedherein. Injectables can be prepared in conventional forms, either asliquid solutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. Suitableexcipients, include by way of example and without limitation, water,saline, dextrose, glycerol or ethanol. In addition, if desired, thepharmaceutical compositions to be administered may also contain minoramounts of non-toxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, stabilizers, solubility enhancers, andother such agents, such as for example, sodium acetate, sorbitanmonolaurate, triethanolamine oleate and cyclodextrins.

Implantation of a slow-release or sustained-release system, such that aconstant level of dosage is maintained (see, e.g., U.S. Pat. No.3,710,795) is also contemplated herein. Briefly, a AhR modulator isdispersed in a solid inner matrix, e.g., polymethylmethacrylate,polybutylmethacrylate, plasticized or unplasticized polyvinylchloride,plasticized nylon, plasticized polyethyleneterephthalate, naturalrubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene,ethylene-vinylacetate copolymers, silicone rubbers,polydimethylsiloxanes, silicone carbonate copolymers, hydrophilicpolymers such as hydrogels of esters of acrylic and methacrylic acid,collagen, cross-linked polyvinylalcohol and cross-linked partiallyhydrolyzed polyvinyl acetate, that is surrounded by an outer polymericmembrane, e.g., polyethylene, polypropylene, ethylene/propylenecopolymers, ethylene/ethyl acrylate copolymers, ethylene/vinylacetatecopolymers, silicone rubbers, polydimethyl siloxanes, neoprene rubber,chlorinated polyethylene, polyvinylchloride, vinylchloride copolymerswith vinyl acetate, vinylidene chloride, ethylene and propylene, ionomerpolyethylene terephthalate, butyl rubber epichlorohydrin rubbers,ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate/vinyl alcoholterpolymer, and ethylene/vinyloxyethanol copolymer, that is insoluble inbody fluids. The agent diffuses through the outer polymeric membrane ina release rate controlling step. The percentage of active agentcontained in such parenteral compositions is highly dependent on thespecific nature thereof, as well as the activity of the agent and theneeds of the subject.

Parenteral administration of the AhR modulators includes intravenous,subcutaneous and intramuscular administrations. Preparations forparenteral administration include sterile solutions ready for injection,sterile dry soluble products, such as lyophilized powders, ready to becombined with a solvent just prior to use, including hypodermic tablets,sterile suspensions ready for injection, sterile dry insoluble productsready to be combined with a vehicle just prior to use and sterileemulsions. The solutions may be either aqueous or nonaqueous.

If administered intravenously, suitable carriers include physiologicalsaline or phosphate buffered saline (PBS), and solutions containingthickening and solubilizing agents, such as glucose, polyethyleneglycol, and polypropylene glycol and mixtures thereof.

Pharmaceutically acceptable carriers used in parenteral preparationsinclude aqueous vehicles, nonaqueous vehicles, antimicrobial agents,isotonic agents, buffers, antioxidants, local anesthetics, suspendingand dispersing agents, emulsifying agents, sequestering or chelatingagents and other pharmaceutically acceptable substances.

Aqueous vehicles include, by way of example and without limitation,Sodium Chloride Injection, Ringers Injection, Isotonic DextroseInjection, Sterile Water Injection, Dextrose and Lactated RingersInjection. Nonaqueous parenteral vehicles include, by way of example andwithout limitation, fixed oils of vegetable origin, cottonseed oil, cornoil, sesame oil and peanut oil. Antimicrobial agents in bacteriostaticor fungistatic concentrations must be added to parenteral preparationspackaged in multiple-dose containers which include phenols or cresols,mercurials, benzyl alcohol, chlorobutanol, methyl and propylp-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride andbenzethonium chloride. Isotonic agents include, by way of example andwithout limitation, sodium chloride and dextrose. Buffers includephosphate and citrate. Antioxidants include sodium bisulfate. Localanesthetics include procaine hydrochloride. Suspending and dispersingagents include sodium carboxymethylcelluose, hydroxypropylmethylcellulose and polyvinylpyrrolidone. Emulsifying agents includePolysorbate 80 (TWEEN® 80). A sequestering or chelating agent of metalions include EDTA. Pharmaceutical carriers also include, by way ofexample and without limitation, ethyl alcohol, polyethylene glycol andpropylene glycol for water miscible vehicles and sodium hydroxide,hydrochloric acid, citric acid or lactic acid for pH adjustment.

The concentration of the pharmaceutically active agent is adjusted sothat an injection provides an effective amount to produce the desiredpharmacological effect. The exact dose depends on the age, weight andcondition of the patient or animal as is known in the art.

The unit-dose parenteral preparations are packaged in an ampoule, a vialor a syringe with a needle. Preparations for parenteral administrationshould be sterile, as is known and practiced in the art.

Illustratively, intravenous or intraarterial infusion of a sterileaqueous solution containing an active agent is an effective mode ofadministration. Another embodiment is a sterile aqueous or oily solutionor suspension containing an active agent injected as necessary toproduce the desired pharmacological effect.

Injectiables are designed for local and systemic administration.Typically a therapeutically effective dosage is formulated to contain aconcentration of at least about 0.1% w/w up to about 90% w/w or more,such as more than 1% w/w of the active agent to the treated tissue(s).The active agent may be administered at once, or may be divided into anumber of smaller doses to be administered at intervals of time. It isunderstood that the precise dosage and duration of treatment is afunction of the tissue being treated and may be determined empiricallyusing known testing protocols or by extrapolation from in vivo or invitro test data. It is to be noted that concentrations and dosage valuesmay also vary with the age of the individual treated. It is to befurther understood that for any particular subject, specific dosageregimens should be adjusted over time according to the individual needand the professional judgment of the person administering or supervisingthe administration of the formulations, and that the concentrationranges set forth herein are exemplary only and are not intended to limitthe scope or practice of the claimed formulations.

The agent may be suspended in micronized or other suitable form or maybe derivatized, e.g., to produce a more soluble active product or toproduce a prodrug or other pharmaceutically acceptable derivative. Theform of the resulting mixture depends upon a number of factors,including the intended mode of administration and the solubility of theagent in the selected carrier or vehicle. The effective concentration issufficient for ameliorating the symptoms of the condition and may beempirically determined.

Lyophilized powders can be reconstituted for administration assolutions, emulsions, and other mixtures or formulated as solids orgels.

The sterile, lyophilized powder is prepared by dissolving an agentprovided herein, or a pharmaceutically acceptable derivative thereof, ina suitable solvent. The solvent may contain an excipient which improvesthe stability or other pharmacological component of the powder orreconstituted solution, prepared from the powder. Excipients that may beused include, but are not limited to, dextrose, sorbital, fructose, cornsyrup, xylitol, glycerin, glucose, sucrose or other suitable agent. Thesolvent may also contain a buffer, such as citrate, sodium or potassiumphosphate or other such buffer known to those of skill in the art at,typically, about neutral pH. Subsequent sterile filtration of thesolution followed by lyophilization under standard conditions known tothose of skill in the art provides the desired formulation. Generally,the resulting solution will be apportioned into vials forlyophilization. Each vial will contain, by way of example and withoutlimitation, a single dosage (10-1000 mg, such as 100-500 mg) or multipledosages of the agent. The lyophilized powder can be stored underappropriate conditions, such as at about 4° C. to room temperature.

Reconstitution of this lyophilized powder with water for injectionprovides a formulation for use in parenteral administration. Forreconstitution, about 1-50 mg, such as about 5-35 mg, for example, about9-30 mg of lyophilized powder, is added per mL of sterile water or othersuitable carrier. The precise amount depends upon the selected agent.Such amount can be empirically determined.

Topical mixtures are prepared as described for the local and systemicadministration. The resulting mixture may be a solution, suspension,emulsions or the like and are formulated as creams, gels, ointments,emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes,foams, aerosols, irrigations, sprays, suppositories, bandages, dermalpatches or any other formulations suitable for topical administration.

The agents or pharmaceutically acceptable derivatives thereof may beformulated as aerosols for topical application, such as by inhalation(see, e.g., U.S. Pat. Nos. 4,044,126, 4,414,209, and 4,364,923, whichdescribe aerosols for delivery of a steroid useful for treatment ofinflammatory diseases, particularly asthma). These formulations foradministration to the respiratory tract can be in the form of an aerosolor solution for a nebulizer, or as a microfine powder for insufflation,alone or in combination with an inert carrier such as lactose. In such acase, the particles of the formulation will, by way of example andwithout limitation, have diameters of less than about 50 microns, suchas less than about 10 microns.

The agents may be formulated for local or topical application, such asfor topical application to the skin and mucous membranes, such as in theeye, in the form of gels, creams, and lotions and for application to theeye or for intracisternal or intraspinal application. Topicaladministration is contemplated for transdermal delivery and also foradministration to the eyes or mucosa, or for inhalation therapies. Nasalsolutions of the active agent alone or in combination with otherpharmaceutically acceptable excipients can also be administered.

These solutions, particularly those intended for ophthalmic use, may beformulated, by way of example and without limitation, as about 0.01% toabout 10% isotonic solutions, pH about 5-7, with appropriate salts.

Other routes of administration, such as transdermal patches, and rectaladministration are also contemplated herein.

Transdermal patches, including iotophoretic and electrophoretic devices,are well known to those of skill in the art. For example, such patchesare disclosed in U.S. Pat. Nos. 6,267,983, 6,261,595, 6,256,533,6,167,301, 6,024,975, 6,010715, 5,985,317, 5,983,134, 5,948,433, and5,860,957.

Pharmaceutical dosage thin's for rectal administration are rectalsuppositories, capsules and tablets for systemic effect. Rectalsuppositories are used herein mean solid bodies for insertion into therectum which melt or soften at body temperature releasing one or morepharmacologically or therapeutically active ingredients.Pharmaceutically acceptable substances utilized in rectal suppositoriesare bases or vehicles and agents to raise the melting point. Examples ofbases include cocoa butter (theobroma oil), glycerin-gelatin, carbowax(polyoxyethylene glycol) and appropriate mixtures of mono-, di- andtriglycerides of fatty acids. Combinations of the various bases may beused. Agents to raise the melting point of suppositories includespermaceti and wax. Rectal suppositories may be prepared either by thecompressed method or by molding. The typical weight of a rectalsuppository is, by way of example and without limitation, about 2 to 3gm.

Tablets and capsules for rectal administration are manufactured usingthe same pharmaceutically acceptable substance and by the same methodsas for formulations for oral administration.

The AhR modulators, or pharmaceutically acceptable derivatives thereof,may also be formulated to be targeted to a particular tissue, receptor,or other area of the body of the subject to be treated. Many suchtargeting methods are well known to those of skill in the art. Suchtargeting methods are contemplated herein for use in the instantcompositions. For non-limiting examples of targeting methods, see, e.g.,U.S. Pat. Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865,6,131,570, 6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975,6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542 and5,709,874.

In some embodiments, liposomal suspensions, including tissue-targetedliposomes, such as tumor-targeted liposomes, may also be suitable aspharmaceutically acceptable carriers. These may be prepared according tomethods known to those skilled in the art. For example, liposomeformulations may be prepared as described in U.S. Pat. No. 4,522,811.Briefly, liposomes such as multilamellar vesicles (MLV's) may be formedby drying down egg phosphatidyl choline and brain phosphatidyl serine(7:3 molar ratio) on the inside of a flask. A solution of an agentprovided herein in phosphate buffered saline lacking divalent cations(PBS) is added and the flask shaken until the lipid film is dispersed.The resulting vesicles are washed to remove unencapsulated agent,pelleted by centrifugation, and then resuspended in PBS.

The AhR modulators or pharmaceutically acceptable derivatives for use inthe methods may be packaged as articles of manufacture containingpackaging material, a AhR modulator or pharmaceutically acceptablederivative thereof, which is effective for modulating the activity of aAhR or for treatment, of one or more symptoms of at least one diseasestate characterized by reduced platelet count and/or platelet functionwithin the packaging material, and a label that indicates that the AhRmodulator or composition, or pharmaceutically acceptable derivativethereof, is used for modulating the activity of AhR for treatment of oneor more symptoms of at least one disease state characterized by reducedplatelet count and/or function.

The articles of manufacture provided herein contain packaging materials.Packaging materials for use in packaging pharmaceutical products arewell known to those of skill in the art. See, e.g., U.S. Pat. Nos.5,323,907, 5,052,558 and 5,033,252. Examples of pharmaceutical packagingmaterials include, but are not limited to, blister packs, bottles,tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, andany packaging material suitable for a selected formulation and intendedmode of administration and treatment.

EXAMPLES

The following examples serve to more fully describe the manner of usingthe invention. These examples are presented for illustrative purposesand should not serve to limit the true scope of the invention.

A. Experimental Procedures

1. iPSC Derivation and Culture Conditions

iPSC derivation was achieved by transduction of the hSTEMCCA lentivirus.The hSTEMCCA lentiviral vector was constructed by ligating cDNA encodinghuman OCT4, KLF4, SOX2, and cMYC into the pHAGE lentiviral plasmid aspreviously described (Somers, A. et al. Generation of transgene-freelung disease-specific human induced pluripotent stem cells using asingle excisable lentiviral stem cell cassette. Stem Cells 28, 1728-1740(2010)). Lentivirus was packaged in 293T cells by co-transfection offive plasmids and were concentrated via a previously publishedultracentrifugation protocol (Sommer, C. A. et al. Excision ofreprogramming transgenes improves the differentiation potential of iPScells generated with a single excisable vector. Stem Cells 28, 64-74(2010); Sommer, C. A. et al. Induced pluripotent stem cell generationusing a single lentiviral stem cell cassette. Stem Cells 27, 543-549(2009)). Peripheral Blood Mononuclear Cells (PBMCs) were used as sourcematerial for iPSC production. Peripheral blood (4 ml) was drawn fromhuman participants into a BD Vacutainer vial (362760). Samples werecentrifuged at 37° C. for 25 minutes at 1800 rcf and the resulting buffycoat was collected in a 15 ml falcon tube. Cells were washed with PBSand counted to ensure that 1×10̂6 cells were isolated for culture. Cellswere resuspended in 2 ml of expansion medium, consisting of QBSF-60(Quality Biological 160-204-101), 50 ng/ml hSCF (R&D 255-SC-010), 10ng/ml hIL-3 (R&D 203-IL-010), 2 U/ml hEPOgen (Amgen), 40 ng/ml hIGF-1(R&D 291-GI-050), 50 ug/ml Ascorbic Acid (Sigma A4403), 100 ug/mlPrimocin (Invivogen ant-pm-2) and 1 μM Dexamethasone (Sigma D4902).After 8-9 days, polybrene was added to the media (5 ug/ml) and thehSTEMCCA lentivirus was added to the culture at an MOI ranging from 1 to10. After 24 hours, the inoculated culture was spun at 2250 g for 90minutes and the polybrene media was discarded. The cells were thenplated onto irradiated Mouse Embryonic Fibroblasts (iMEFs) and culturedfor roughly 15 days in “iPSC media” that includes DMEM F12 (Invitrogen11330057) 10 ng/ml bFGF (R&D 233-FB-025) 1 ng/ml Rho Kinase Inhibitor(Cayman Chemical 10005583) 20% Knock-out Replacement Serum (KOSR)(Invitrogen 10828028) and 100 ug/ml Primocin. Clones were then pickedand expanded into long-term culture.

2. Directed Differentiation of iPSCs into Mesoderm Cell Fate

High passage iPSCs were plated onto matrigel coated 6-well plates iniPSC media conditioned on iMEFs for 24 hours and supplemented with 2ng/ml Rho Kinase Inhibitor and 20 ng/ml bFGF. After two days, iPSC mediawas replaced with Mesoderm D0-1 media: RPMI (Invitrogen A1049101)supplemented with 5 ng/ml hBMP-4 (R&D 314-BP-010), 50 ng/ml hVEGF (R&D293-VE-010), 25 ng/ml hWnt3a (R&D 287-TC-500) and 10% KOSR. At Day 2,Mesoderm D0-1 media was replaced with Mesoderm D2 media: RPMIsupplemented with 5 ng/ml hBMP-4, 50 ng/ml hVEGF, 20 ng/ml bFGF and 10%KOSR. Mesoderm D3 media consisted of the following: StemPro 34(Invitrogen 10639011), 5 ng/ml hBMP-4, 50 ng/ml hVEGF, and 20 ng/mlbFGF. Mesoderm media for days 4 and 5 consisted of: StemPro 34, 15 ng/mlhVEGF, and 5 ng/ml bFGF. Day 6 mesoderm media: 74% IMDM (Invitrogen12330061), 24% Hams F12 (Mediatech 10-080-CV), 1% B27 supplement(Invitrogen 12587-010), 0.5% N2-supplement (Invitrogen 17502-048), 0.5%BSA (Sigma A3059), 50 ng/ml hVEGF, 100 ng/ml bFGF, 100 ng/ml hSCF (R&D255-SC-010), 25 ng/ml hFlt3 Ligand (R&D 308-FKN-005). Day 7 media: 74%IMDM, 24% Hams F12, 1% B27 supplement, 0.5% N2-supplement, 0.5% BSA, 50ng/ml hVEGF, 100 ng/ml bFGF, 100 ng/ml hSCF, 25 ng/ml hFlt3 Ligand, 50ng/ml hTPO (Genentech G140BT), 10 ng/ml IL-6 (R&D 206-IL-010), 0.5 U/mlhEPOgen and 0.2 uM 6-formylindolo[3,2-b]carbazole (FICZ) (Santa CruzSC300019). After Day 7, 0.5 ml of Day 7 media was added to the culturedaily without aspirating the media from the previous day. All base mediamixes included 2 mM L-Glutamine (Invitrogen 25030081), 4×10̂-4MMonothioglycerol (Sigma M1753), 100 ug/ml Primocin, and 50 ug/mlAscorbic Acid. Cells in suspension were collected and assayed at Days10-13 or split for long-term culture.

3. Lentiviral Vector Generation and Application

PCR primers were designed to amplify the MMTV-DRE (mouse mammary tumourvirus/dioxin response element region from murine CY1A1 gene) promoterregion from AHR activity reporter construct pGudLuc1.1, with integratedSpel and NotI cut sites at the 5′ and 3′ ends respectively. Therestriction enzyme digested PCR product was then inserted into thepHAGE2 lentiviral Eflα-dsRed(NLS)-IRES-ZsGreen plasmid and the pHAGE2lentiviral Eflα-destabilized ZsGreen by excision of the Eflα promoterand ligation of the Spel and NotI digested MMTV-DRE. Additionally, theAHR repressor was cloned into the pHAGE2 lentiviralEfla-dsRed(NLS)-IRES-ZsGreen. Primers were designed to amplify thef.heteroclitus AHRR coding region from an HPV422-based construct, withNotI/BamHI cut sites incorporated at the 5′ and 3′ sites respectively.The dsRed (NLS) insert was excised and digested AHRR was ligated intothe aforementioned vector.

VSV-G pseudotyped lentiviral particles were packaged and concentrated aspreviously published. (Murphy, G. J., Mostoslaysky, G., Kotton, D. N. &Mulligan, R. C. Exogenous control of mammalian gene expression viamodulation of translational termination. Nat Med. 12, 1093-1099. Epub2006 August 1096. (2006)) Cells were infected overnight and subsequentdsRed and ZsGreen gene expression was monitored by fluorescencemicroscopy and flow cytometry as indicated in the text.

4. AHR Small Molecule Competition Assays

6-formylindolo[3,2-b]carbazole (FICZ), an AHR small molecule agonist,and CH223191, an AHR competitive inhibitor were used for these assays.CH223191 was added to mesoderm cultures at Day 6 at 5 uM (lx) and 2.5 uM(0.5×). 0.2 uM FICZ was added to cultures at Day 7 and media was addeddaily. DMSO was used as a vehicle control.

5. Quantitative RT-PCR

RNA was extracted using the RNeasy kit (Qiagen) according to themanufacturer's instructions and DNase treated using the DNA-free kit(Ambion AM1906). Reverse transcription into cDNA was performed using theHigh Capacity cDNA Reverse Transcription Kit (Applied Biosystems4368814). Quantitative (real time) PCR amplification of cDNA wasperformed using Taqman probes for AHR (Hs00169233_m1), CYP1B1(Hs002382916_s1), HBA (Hs00361191_g1), HBB (Hs00758889_s1), HBG(Hs01629437_s1), vWF (Hs00169795_m1), PF4 (Hs00427220_g1), NF-E2(Hs00232351 m1) and CD62P (Hs00927900_ml) and run on the AppliedBiosystems StepOne machine. Relative gene expression was normalized toB-actin (Hs99999903_m1).

6. Flow Cytometry

Roughly 10̂5 cells were collected, spun, and re-suspended in 0.5% BSA inPBS. Samples were incubated for 30 min at ambient temperature with humanantibodies including CD41a-FITC (BD 555466), CD235-PE (BD 555570),CD71-FITC (BD 555536), washed and spun at 3300 rpm for 7 min, andre-suspended in 0.5% BSA in PBS with 1 ug/ml Propidium Iodide. Sampleswere run on a BD FACScalibur using Cellquest Pro software and analyzedvia FloJo 8.7. For ploidy analysis, cells were treated with 1.5% NP-40(Boston Bioproducts P-872) and 62.5 ug/ml Propidium Iodide in PBSimmediately before FACScalibur interrogation. For murine bone marrow,samples were first incubated for 5 min at ambient temperature withmurine conjugated antibody CD16/32 (BD 553142) before a 30 minincubation with c-Kit-PE (BD 553355), CD41a-FITC (BD 553848), Ter119-PE(BD 553673). For cell viability assays, 2-3 x 10̂5 cells were collected,re-suspended in 8.8 ug/ml Hoecsht 33342 in PBS supplemented with 5% FBS.Samples were then incubated in the dark at 37° C. for 15 min, washed,and re-suspended in 1 ug/ml Propidium Iodide in 5% FBS. Samples were runon an LSR-II machine with FACSDiva software and analyzed via FloJo 8.7.

7. Gene Expression Analysis

The data analyzed correspond to the RMA-processed, batch-normalized,Affymetrix® expression profiles downloaded from the dMap website(www.broadinstitute.org/dmap). This includes the expression levels of8968 Entrez-annotated genes across 212 experiments representing 15distinct populations (38 sub-populations) of hematopoietic cells. Thedata was projected onto the space of 37 manually curated AhR targets,plus AhR itself, and 72 experiments corresponding to 5 populations (11sub-populations), defining the HSC-to-Mk/erythroid differentiation path.The genes were sorted based on hierarchical clustering with 1-Pearsoncorrelation as the distance metric, and average linkage as theagglomeration rule (Eisen, M. B., Spellman, P. T., Brown, P. O. &Botstein, D. Cluster analysis and display of genome-wide expressionpatterns. Proc Natl Acad Sci USA 95, 14863-14868 (1998).) (FIG. 1 a).The normalized expression level of AhR within each cell population(sub-population) was computed and visualized by means ofbox-and-whiskers plots (FIG. 1 b). For each population, the plot reportsthe median (thick mid line), the middle half (the box), and theInterquartile Range (IQR, the distance between the “whiskers”) of thedistribution of AhR values. The difference in the expression level ofAhR among cell populations was tested by standard analysis-of-variance(anova).

8. Statistical Analysis

Results are presented as the mean±the standard deviation of experimentsperformed in triplicate. Statistical significance was confirmed usingthe Student's t-test.

9. In Vivo Studies

C57B16 mice were injected daily intraperitoneally with FICZ suspended invegetable oil using a weekly dose escalation scheme (Week 1: 1 mg/kg;Week 2: 2 mg/kg; Week 3: 4 mg/kg). Blood cell counts were assayed byHemavet quantification of peripheral blood bleeds, at all 3 time points(Day 7, 14, and 21). Following the 3 week time point, mice weresacrificed and livers and spleens harvested for quantitative RT-PCRanalyses.

Example 1 Analysis of Human Hematopoietic Cell Differentiation GenomicMapping (dMap) Data

As a roadmap for assessing the possible role of the AhR receptor inhematopoietic cells, we analyzed the “dMap” dataset(www.broadinstitute.org/dmap) (Novershtern, N. et al. Denselyinterconnected transcriptional circuits control cell states in humanhematopoiesis. Cell 144, 296-309 (2011)) a publicly available compendiumof expression profiles from 71 distinct purified populations of humanhematopoietic cells. For our purposes, we focused on theHSC-to-Mk/erythroid differentiation path, and we analyzed the expressionof a manually curated list of putative AhR targets. Hierarchicalclustering was carried out to evaluate the co-expression patterns of AhRand its targets. This analysis revealed up-regulated Ahr mRNA expressionin primitive stem cells, from the HSC to the MEP cell stage (FIG. 1).Erythroid cells clustered into 2 groups of cells with either up- ordown-regulated Ahr. Ahr levels were consistently up-regulated in Mks.The levels of approximately 14 putative AhR target genes, includingseveral of significant import to stem cells (e.g., c-myc, EGR1, andALDHA1) were coordinately regulated with Ahr levels. Other importanthematopoietic-specific genes such as NFE2, a critical regulator of boththe erythroid and Mk lineages, also displayed coordinated differentialexpression with Ahr. These results indicated that Ahr expression isevident in hematopoietic progenitor cells and suggested that AhR mayplay a role in the development of human bipotential MEPs. These data,which clearly demonstrate AhR expression throughout the humanhematopoietic system, allowed us to formulate a hypothesis that AhRactivation could be used in an in vitro system to greatly enhance anddirect the production and differentiation of hematopoietic progenitorcells.

Example 2 Production of Megakaryocyte-Erythroid Progenitors (MEPs) FromInduced Pluripotent Stem Cells (iPSCs)

This example demonstrates the feeder-free, chemically defined productionof megakaryocyte-erythroid progenitors (MEPs) from induced pluripotentstem cells (iPSCs), and shows that the cells express definitive markersof both the megakaryocyte and erythroid lineages. We sought to develop anovel, feeder-free, chemically-defined system for the production ofhematopoietic progenitor cells from human iPSCs that would not bebeholden to the use of stromal cell lines or xenogeneic agents, andwould result in the ability to produce large numbers of clinicallyrelevant, high purity hematopoietic cells. The approach employed in thedevelopment of this platform follows the roadmap provided by thedeveloping embryo. Since ESC and iPSC resemble pluripotent,undifferentiated cells of the early blastocyst embryo, the signalsactive in the early embryo were harnessed to direct the differentiationof ESC and iPSC in vitro. Due to the known variability in the formationof human embryoid bodies (Bratt-Leal, A. M., Carpenedo, R. L. &McDevitt, T. C. Engineering the embryoid body microenvironment to directembryonic stem cell differentiation. Biotechnology progress 25, 43-51(2009)), our protocol utilized a 2D culture system optimized to producebipotential hematopoietic progenitor cells within 10-13 days (FIG. 2A).A key element in this platform was the addition of a strong AhR ligand,FICZ, on day 7. The timeframe to generate MEPs is significantly shorterthan that noted in previously described protocols (Takayama, N. et al.Generation of functional platelets from human embryonic stem cells invitro via ES-sacs, VEGF-promoted structures that concentratehematopoietic progenitors. Blood 111, 5298-5306 (2008); Gekas, C. &Graf, T. Induced pluripotent stem cell-derived human platelets: one stepcloser to the clinic. The Journal of experimental medicine 207,2781-2784 (2010)) and requires no fractionation or further manipulationof the cells. In this system, differentiating iPSC produce anendothelial cell-based adherent layer from which non-adherenthematopoietic cells emerge beginning at Day 7 (FIG. 2A). As judged byimmunophenotyping at Day 15, greater than 50% of these cells co-expressCD235-Glycophorin A (erythroid lineage) and CD41 (Mk lineage) suggestingthat bipotential MEPs had been generated (FIG. 2B). In comparison toundifferentiated iPSCs, these cells also upregulate globin geneexpression and express a series of hallmark Mk markers (FIG. 2D).Furthermore, through the use of erythroid specification media containingEPO or Mk specification media containing TPO, iPSC-derived MEPs undergoa final fate choice in order to become either mature erythrocytes or Mks(FIG. 2C).

Example 3 The Aryl Hydrocarbon Receptor (AhR) Agonist FICZ Allows forthe Exponential Expansion of iPSC-Derived MEPs

Translation of iPSC technology to clinical applications has beenhindered by the inability to produce sufficient, clinically relevantquantities of cells. Even for basic research studies, the numbers andquality of hematopoietic cells that can be produced through the directeddifferentiation of iPSC can be limiting (Chang, K. H., Bonig, H. &Papayannopoulou, T. Generation and characterization of erythroid cellsfrom human embryonic stem cells and induced pluripotent stem cells: anoverview. Stem Cells Int. 2011, 791604. Epub 792011 Oct 791626. (2011)).Here, we demonstrate that the AhR agonist FICZ has the ability to allowfor the exponential expansion of iPSC-derived MEPs. In comparison tountreated control samples, FICZ-treated day 30 MEPs demonstratesignificantly less cell death as judged by propidium iodide staining andHoecsht dye exclusion allowing for the exponential expansion of thepopulation (FIGS. 3A and B). As demonstrated in these plots,FICZ-treated cells have both increased viability with fewer cellsundergoing apoptosis. Day 15 MEPs were also grown with or without thepresence of FICZ and growth rates for each population were calculated.In contrast to untreated cells, FICZ treated MEPs demonstratedlogarithmic expansion over a 2 week growth period (FIG. 3C).

In a subsequent experiment, N-ethyl-N-nitrosourea (EDU) incorporation inday 30 MEPs was used to compare proliferation of FICZ-treated MEPS andcontrol untreated MEPs. EDU is a labeling chemical that intercolatesinto the DNA of a cell and allows for the explicit tracking ofproliferation. As shown in (FIG. 3D), day 30 MEPs that have been treatedwith the AhR agonist FICZ are far more proliferative than untreatedcells.

Example 4 AhR Agonists Induce CYP1B1 Target Gene Expression in HumaniPSCs and MEPs

To characterize AhR expression and functionality in bothundifferentiated iPSCs and directly differentiated MEPs, AhR proteinlevels were determined via Western blot in these populations. AhRreceptor was robustly expressed in day 30 and day 60 MEPs (FIG. 4A).However, AhR protein was not detected by western blotting in iPSCpopulations, and we postulated that AhR was expressed at extremely lowlevels in iPSCs, i.e., below the level of detectability with theantibodies used for Western blots. To test this hypothesis, the abilityof FICZ to induce a prototypic AhR-target gene, CYP1B1, in iPSCs or, asa positive control, MEPs was assessed by quantitative RT-PCR. Bothundifferentiated iPSCs and directly differentiated MEPs showedstatistically significant increases in CYP1B1 expression followingtreatment with FICZ strongly suggesting that the AhR receptor is indeedexpressed in these cells (FIG. 4B). Notably, these cell population alsowere responsive to other AhR agonists including the prototypicenvironmental AhR ligand, 2,3,7,8-tetrachlorodibenzo(p)dioxin (TCDD)(FIG. 13).

Example 5 AhR Mediates the Expansion and Specification of BipotentialHematopoietic Progenitors

To quantify AhR transcriptional activity, presumably mediated by anendogenous AhR ligand, we cloned a human AhR-responsive promoter (67,68) into a lentivirus reporter vector that encodes for either nuclearlocalized dsRed and ZsGreen or luciferase and ZsGreen (FIG. 5A). Thesedual gene “AhR reporters” allow for normalization of transductionefficiency, negate any effect of auto-florescence, and allow for thequantification of AhR transcriptional activity. Day 30 MEPs weretransduced with reporter lentivirus or mock infected at a multiplicityof infection (MOI) of 10 and grown in basal medium containing 0.2 μMFICZ for 72 hours. MEPs were then subjected to three different growthconditions in order to assess the activity of AhR in this population ofcells: 1) The steady state condition consisting of 0.2 μM FICZ; 2) anincrease in FICZ concentration to 0.4 μM; or 3) 0.2 μM FICZ plus 5 μM ofthe known AhR inhibitor CH223191 (Kim, S. H. et al. Novel compound2-methyl-2H-pyrazole-3-carboxylic acid(2-methyl-4-o-tolylazo-phenyl)-amide (CH-223191) prevents2,3,7,8-TCDD-induced toxicity by antagonizing the aryl hydrocarbonreceptor. Mol Pharmacol. 69, 1871-1878. Epub 2006 March 1815. (2006)).In contrast to the mock infected MEPs, the AhR reporter-infectedpopulation displayed a modest increase in dsRed expression suggestingthat the Dioxin Responsive Element in the reporter was beingtransactivated in the MEPs (FIG. 5B). When the reporter infected MEPswere subjected to an increased amount of the AhR agonist FICZ (0.4 μM),a significant increase in DsRed expression was noted, demonstrating thatFICZ is capable of transactivating the AhR receptor in primary,iPSC-derived, directly differentiated MEPs (FIG. 5B, 5C). This resultwas also confirmed visually via immunoflourescence microscopy withZsGreen+ cells only noted in the MEPs treated with 0.4 μM FICZ (FIG.5D). Importantly, when the reporter-infected populations were subjectedto growth medium containing 5 μM of the known AhR inhibitor CH223191, asignificant decrease (below the level of expression in the mock infectedpopulations) was noted in DsRed expression further demonstrating thatFICZ-mediated transcriptional activity is mediated through the AhRreceptor in iPSC-derived, directly differentiated MEPs (FIG. 5B). Theseresults were confirmed quantitatively using a lentiviral backbone thatencoded luciferase (FIG. 5C)

In order to determine whether FICZ-mediated transactivation of the AhRreceptor was responsible for the exponential expansion of iPSC-derivedMEPs, the previously described Hoecsht/Propidium Iodide apoptosis assaywas performed using the known AhR inhibitor CH223191. As previouslyshown in FIG. 3, fewer cells stained with propidium iodide after FICZtreatment (e.g., 15.5% vs. 8.09%) (FIG. 5E). In contrast, when cellswere pre-treated for 24 hours with 5 μM CH223191, the percentage of PI⁺cells was approximately the same in vehicle or FICZ-treated cultures. Nosignificant expansion of the CH223191+FICZ-treated cells was noted.Interestingly, when a lower dose of the inhibitor was used (2.5 μM) topre-treat the cells before the addition of FICZ, the cells were stillcapable of expansion suggesting that agonist/antagonist interaction andbinding of the AhR receptor in the iPSC-derived, directly differentiatedMEPs is dose dependent (FIG. 5E). The efficacy of the CH223191 wasconfirmed by its ability to block CYP1B1 induction as assayed by qRT-PCR(FIG. 5F).

Example 6 Continuous AhR Activation Allows for Red Blood Cell MaturationWhile Inhibition/Antagonism Promotes Megakaryocyte Development

Previous studies suggest that the AhR may play a critical role inhematopoietic cell development and function, possibly including growthand differentiation of hematopoietic stem cells. Having shown that AhRactivation results in exponential expansion of MEP populations (FIG. 3),we then were in a position to determine if the AhR also contributes toMEP differentiation into RBC or megakaryocytes. Given a proposed, butnot yet clearly defined role of the AhR during hematopoieticdevelopment, we conducted a series of experiments to elucidate the roleof AhR in bipotential hematopoietic progenitor cells and their resultantprogeny.

In our previous studies in which we noted exponential expansion ofiPSC-derived MEPs (FIG. 3), the AhR-mediated effect that we notedallowed us to culture cells for extended periods of time (>120 days).Immunophenotyping of MEPs maintained in feeder-free conditions in thepresence of FICZ revealed a progressive erythroid specification andmaturation under continuous AhR agonism. As demonstrated in FIG. 6A, themajority of early passage (Day 15) iPSC-derived MEPs expressed CD71(transferrin receptor) with a small portion of the cells also expressingCD235 (glycophorin A) suggesting an immature red cell phenotype. Underprolonged exposure to FICZ (30 days), these cells began to down regulateexpression of CD71 and a larger percentage of cells expressed CD235suggesting a more mature phenotype (FIG. 6A). As these cells continuedto specify to the erythroid lineage under basal growth conditions withAhR agonism, maturation continued resulting in a more homogenouspopulation that contained few megakaryocyte-lineage cells. Thispopulation was almost entirely CD235 ⁺ (FIG. 6B). These populations ofiPSC-derived erythrocytes demonstrated functional maturity as assessedby their ability to respond to hypoxic conditions (FIG. 6C) and toproduce hemoglobin (FIG. 6D). For example, when cultured under lowoxygen (5% O₂) to simulate stress erythropoiesis, cells began to displayhallmark characteristics of maturing erythroblasts including a reductionin cell size and the condensation of chromatin within the nuclei of thecells (FIG. 6C). More strikingly, when cells were centrifuged, brightred pellets were noted suggesting the production of hemoglobin. Whenadditional EPO was added to the cultures, still more red cells werenoted in the pellets (FIG. 6D).

As the default pathway in our system seemed to allow for thespecification and maturation of iPSC-derived MEPs into the red celllineage under AhR agonism, we hypothesized that further AhR modulationwould allow for the development of the alternative Mk lineage. To testthis hypothesis, we conducted studies that allowed for AhR antagonismusing both small molecule inhibition of the receptor and forcedexpression of an AhR repressor protein. In the first set of experiments,the known AhR antagonist CH2223191 was added to day 30 MEP populationsgrown in basal cytokine conditions. In contrast to vehicle-treatedcontrol populations in which virtually no CD41 positivemegakaryocyte-lineage cells were noted, MEPs treated with the AhRinhibitor produced a small but defined population of CD41⁺megakaryocyte-lineage cells (FIG. 6E). In a second set of experiments,we constructed and utilized a lentiviral vector that encoded an AhRrepressor element (AhRR) along with a ZsGreen reporter (FIG. 6F). Inseveral of our studies, this AhRR element potently and specificallyinhibited either baseline or AhR agonist-induced AhR transcriptionalactivity (Hahn, M. E., Allan, L. L. & Sherr, D. H. Regulation ofconstitutive and inducible AHR signaling: complex interactions involvingthe AHR repressor. Biochem Pharmacol 77, 485-497 (2009); Evans, B. R. etal. Repression of aryl hydrocarbon receptor (AHR) signaling by AHRrepressor: role of DNA binding and competition for AHR nucleartranslocator. Mol Pharmacol 73, 387-398 (2008)). In contrast tomock-infected MEPs which were transduced with a constitutively activeZsGreen reporter only, cells infected with the AhRR lentivirus produceda significant number of CD41⁺ megakaryocyte-lineage cells (FIG. 6G).Interestingly, while the AhRR-transduced populations were capable ofproducing megakaryocyte-lineage cells, they also contained fewer CD235⁺cells, suggesting that AhR antagonism in iPSC-derived MEPs initiated atranscriptional switch from erythroid to megakaryocyte-lineagespecification (FIG. 6G). To further study the megakaryocyte-lineagecells produced via AhR antagonism in iPSC-derived MEPs, a discontinuousBSA gradient (0, 1.5, 3%) was used to isolate maturing Mks. Remarkably,large, CD41⁺, polyploid Mks were produced following the suppression ofAhR activity via AhRR overexpression (FIG. 6H). These cells demonstratedhallmark characteristics of mature Mks including the ability toendoreplicate to 8N and 16N (FIG. 6I) and the presence of proplateletextrusions at the surface of the cells (FIG. 6H). Furthermore, incontrast to mock-infected controls, large, AhRR-expressing Mks werenoted in both early and later stage MEP cultures (FIG. 6J).

FIG. 7 presents a mechanistic diagram of the role of AhR agonism in thedifferentiation and expansion of MEPs and the roles of AhR agonism andantagonism in the differentiation of RBCs and megakaryocytes from MEPs.

Example 7 Characterization of iPSC-Derived RBCs

Expression of genes involved in reprogramming of iPSCs and genesinvolved in RBC differentiation were analyzed to further characterizeiPSC-derived RBCs made according to the methods of Example 6. Theresults show that embryonic genes (including those such as Oct4, Sox2,and Nanog that are responsible for the reprogramming process aredownregulated as cells are directly differentiated into RBCs (FIG. 8Aand data not shown).

At days 15 and 30 of erythroid specification in this directeddifferentiation strategy the cells exhibit a complementary upregulationof genes of critical import to the RBCs (FIG. 8B).

By microarray analysis, Day 15 and Day 30 iPSC-derived RBCs upregulate apanel of hemoglobins (alpha 1 and gamma 2), and other genes involved inthe regulation of the erythroid-lineage (p45, NFE2; c-Myb; Kruppel-likefactor 1, KLF-1; alpha hemoglobin stabilizing protein, AHSP; and CD235,Glycophorin A. In addition, BCL-11A is downregulated upondifferentiation which is commensurate with erythroid maturation.

Example 8 Characterization of iPSC-Derived RBCs

In normal hematopoietic development, embryonic globins (epsilon andzeta) are expressed early on in development and downregulated pre-birth.Alpha globin is expressed at high levels both pre and post birth. Fetalhemoglobin (gamma) is expressed at high levels pre-birth, and is rapidlydownregulated (with less than 5% expressed by 5 years of age. Adultglobin (beta) is expressed reciprocally with fetal hemoglobin, and isthe predominant form of hemoglobin expressed in adult cells.Importantly, the ability to increase fetal hemoglobin in an adultameliorates the symptomology of sickle cell anemia.

We utilized mass spectrophotometric analyses to study the types ofglobins that are being produced by iPSC-derived RBCs using the methodsof this disclosure. In FIG. 9, results of the analyses of wholeperipheral blood of a control patient (FIG. 9A) and a patient sufferingfrom sickle cell disease (FIG. 9B). Clear peaks for alpha globin, gammaglobin, and beta globin (adult globin) are evident. In the sickle cellpatient the mutation that produces sickle cell (hemoglobin S) is clearlyvisible (FIG. 9B). The results of a similar analysis of day 30iPSC-derived RBCs is shown in FIG. 10. The predominantly expressedproteins are the globins. The cells clearly express alpha robustly.Interestingly, the cells are apparently at an embryonic/fetal stage ofdifferentiation, in that they express both embryonic globins (epsilonand zeta) as well as fetal (gamma; there are two peaks here as there aretwo isoforms of gamma), but no adult globin (beta). iPSC-derived RBCshave not been previously analyzed in this manner, and these massspectrophotometric analyses provide evidence that the cells express theappropriate genes at the protein level.

Example 9 iPSC-Derived RBCs Respond to HbF Inducers

The ability to increase fetal hemoglobin in an adult ameliorates thesymptomology of sickle cell anemia. Hydroxyurea (HU) is the onlyFDA-approved drug that does this, presumably by initiating stresserythropoiesis (this is a process by which new red cells are rapidlybirthed under stress; as they are recently emerged RBCs they express abit more fetal hemoglobin). As discussed above, our iPSC-derived RBCsalready express fetal hemoglobin, so the question arose as to whether ornot the cells would be responsive to HU (and could therefore be used asa patient-specific screening platform for novel inducers of HbF). Asshown in FIG. 11, exposure of iPSC-derived RBCs to 0.5 μM HU causes a4-fold increase in expression of fetal hemoglobin (gamma) (HBG in FIG.11). This data illustrates the fact that the cells are indeed responsiveto therapeutic doses of HU.

Example 10 AhR Agonism Promotes MEP Production and Expansion in MurineBone Marrow

To determine if AhR agonism would result in MEP production and expansionfrom bone marrow precursors (as opposed to iPSCs) in a murine system,red cell-depleted, bone marrow from C57BL/6 mice was cultured for 3 daysin the presence or absence of vehicle or 0.2 μM FICZ. Remarkably, incontrast to vehicle-treated controls, distinct populations of primary,CD41⁺/Ter119⁺ MEPs were noted in the cultures following just 3 days ofFICZ treatment (FIG. 12).

Example 11 iPSC-Derived Mks Upregulate Key Megakaryocyte-Specific Genes

To further characterize iPSC-derived Mks made according to methods ofthis disclosure Quantitative PCR analysis was performed followingpurification using a discontinuous BSA gradient. iPSC-Mks, created usingAhR anatagonism, express a series of hallmark and characteristic MKmarkers. (FIG. 14). By quantitative PCR analysis, these cells expresshallmark and characteristic MK markers such as CD62P (P-selectin),Platelet Factor 4 (PF4), GPIIb, and GPV.

Example 12 iPSC-Derived Mks Produce Functional Platelets

Flow cytometry was used to compare platelets from whole blood andiPSC-derived platelets. The results reveal that iPSC-derived plateletsare remarkably similar to those derived from whole blood. The FSC vs.SSC profile is extremely similar, and iPSC-derived platelets express thehallmark platelet markers GPIX, GPIb, and P-Selectin. (FIG. 15).

Example 13 The AhR Agonist FICZ is Active In Vivo and Results inIncreased Platelet Counts in Normal Mice

To determine if FICZ treatment or whole animals would affect RBC orplatelet production, C57B16 mice were injected daily intraperitoneallywith FICZ suspended in vegetable oil using a weekly dose escalationscheme (Week 1: 1 mg/kg; Week 2: 2 mg/kg; Week 3: 4 mg/kg). In contrastto mock-injected mice or mice injected with vehicle only, mice injectedwith FICZ showed increased platelet counts, as assayed by Hemavetquantification of peripheral blood bleeds, at all 3 time points (Day 7,14, and 21) (FIG. 16A). Interestingly, a mouse that was immediatelyexposed to higher doses of FICZ (4 mg/kg) and did not undergo week 1escalation demonstrated a more immediate and prolific platelet response.None of the mice in the study showed significant variations in eitherwhite blood cell (WBC) or red blood cell (RBC) counts (not shown).Following the 3 week time point, mice were sacrificed and livers andspleens harvested. Quantitative RT-PCR analyses for CYP1B1 target geneexpression revealed robust upregulation in the liver and spleen of FICZtreated animals confirming that we had reached biologically meaningfulFICZ doses in vivo (FIGS. 16B and 16C).

Example 14 AhR Inhibition in iPSC Cells

iPSC cell lines were produced that allow selective downregulateion ofexpression of AhR using a molecular approach. FIG. 17 shows theconstruct used, which contains a short hairpin RNA (RNAi) for AhR whichis expressed when the cells are treated with a doxicycline inducer. Ared florescent reporter is also turned on to track the expression. Thetop panel of FIG. 17 shows that the RNAi can be turned on in theundifferentiated cells and at Day 5 of the differentiation.

The RNAi for AhR was activated in end stage MKs made using thetechniques of this disclosure. The result is an increase in productionof proplatelets compared to control cells expressing a scrambled RNAisequence (SCR). Images of the fluorescent reporter are shown in FIG. 18(top) and the dramatic increase in proplatelet formation is presentedgraphically (FIG. 18 (bottom)).

Discussion:

Our results indicate that AhR has a physiological and functional role innominal hematopoietic development, and that modulation of the receptorin bi-potential hematopoietic progenitors can direct cell fate. Wedemonstrate a novel methodology for the directed differentiation ofpluripotent stem cells in serum and feeder-free defined cultureconditions into MEPs capable of final specification into Mks and/orerythroid-lineage cells.

As a starting point for these studies, we utilized human hematopoieticcell differentiation genomic (dMap) array data as a roadmap forassessing the possible role of AhR in hematopoietic cells. Theseanalyses were a powerful tool that suggested that the AhR plays animportant role in blood cell development and were consistent withprevious studies and the considerable amount of data presented here.

Although several teams have published proof-of-principle examples forthe derivation of hematopoietic cells from ESC and iPSC these protocolsare technically demanding and result in the production of limitednumbers of cells. Our conceptual approach has been to mimic the naturalsequences of development in vitro in order to derive the range andnumber of cell types needed for the creation of a robust iPSC-basedplatform. This protocol utilizes a relatively simple 2D culture approachand eliminates the need for embryoid body formation, often a problematicstep when using human pluripotent stem cells. Furthermore, this protocolis short (˜10 days), completely chemically defined, and requires noxenobiotic feeder cells or growth factors thereby making GMP productionand clinical translation feasible.

Importantly, we have also found that the use of a non-toxic arylhydrocarbon receptor agonist in our directed differentiation schemedramatically increases the number of MEPs and resultant cells. This isan extremely important finding in that traditionally, the evolutionarilyconserved AhR has been studied for its role in environmentalchemical-induced toxicity, and in our system it is shown to beintricately involved in the growth and the differentiation of at leasttwo crucial blood cell types. Following the addition of the potent AhRligand FICZ to our cultures, we observed exponential expansion of MEPsfrom a few thousand to a billion cells in a few weeks. Importantly, therole of AhR in the MEP population was confirmed using a highly specificAhR inhibitor. This logarithmic expansion of cells appears to be afunction of decreased cell death and is consistent with previous studieswhich suggest that the AhR can control apoptosis.

Interestingly, FICZ, the AhR ligand utilized throughout this work, is aphoto-metabolite of tryptophan originally described by Rannug andcolleagues (Rannug, U. et al. Structure elucidation of twotryptophan-derived, high affinity Ah receptor ligands. Chem Biol. 2,841-845. (1995)). Based on previous studies demonstrating the ubiquityof FICZ (Wincent, E. et al. The suggested physiologic aryl hydrocarbonreceptor activator and cytochrome P4501 substrate6-formylindolo[3,2-b]carbazole is present in humans. J Biol Chem 284,2690-2696 (2009)) and taken together with our data demonstrating the invivo activity of this ligand, it is not inconceivable that FICZ plays arole in regulating hematopoiesis in vivo, possibly with other endogenousAhR ligands also playing a role. The ability to expand MEPs with an AhRligand also suggests that blood cell development may be affected by avariety of environmental ligands.

In addition to allowing for the exponential expansion of MEPs, ourresults indicate that AhR modulation is also involved in the furtherspecification of both the erythroid and Mk lineages with AhR agonismpermissive to the development of erythroblasts and antagonism or downregulation of AhR leading to Mk development. Although erythropoietin(EPO) and thrombopoietin (TPO) are the major drivers in RBC and plateletdevelopment, the data presented herein points to a cytokine-independentrole for AhR in the development and specification of these lineages.

During the course of our studies we derived putative progenitors knownto express markers of both the Mk and erythroid lineages. A particularlystriking outcome of our experiments is the development of a simpleprotocol for the rapid and highly efficient derivation of putative MEPswhich expand exponentially under AhR agonism. In addition to the abilityto answer basic biological questions concerning hematopoieticdevelopment, a useful outcome for this work will be the utilization ofthis in vitro platform for the clinically relevant production of bloodproducts. Blood transfusion is an indispensable cell therapy, and thesafety and adequacy of the blood supply are national and internationalconcerns. An iPSC-based system, such as the one described here in whichsufficient numbers of cells can be produced, could allow for red bloodcell and platelet transfusion without problems related toimmunogenicity, contamination, or supply. Furthermore, the ability toproduce both populations of cells from a single source, and the factthat both platelets and mature RBCs contain no nuclear genetic materialdecreases safety concerns with the use of iPSC-derived cells and pavesthe way for clinical translation.

In conclusion, we present the development of a novel, chemicallydefined, and feeder-free methodology for the production of iPSC-derivedhematopoietic cells. This methodology allows for exponentially greaterproduction of RBCs and platelets in comparison to existing methodologiesand relies on the first of its kind definition of the role of the AhRreceptor in nominal hematopoietic development using specialized ligandsin hematopoietic progenitor cells.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1-116. (canceled)
 117. A method of making a platelet, comprising: differentiating a pluripotent stem cell into a myeloid-erythroid progenitor cell (MEP) in culture in the presence of an aryl hydrocarbon receptor (AhR) agonist; culturing the MEP under conditions sufficient to make a Mk; and culturing the Mk under conditions sufficient for differentiation of a platelet from the Mk.
 118. The method of claim 117, wherein the conditions sufficient to make a Mk comprise culturing the MEP in the presence of an AhR modulator.
 119. The method of claim 118, wherein the AhR modulator is an AhR antagonist.
 120. The method of claim 117, wherein the conditions sufficient to make a Mk comprise culturing in megakaryocyte specification media.
 121. The method of claim 120, wherein the conditions sufficient to make a Mk further comprise culturing in the presence of an AhR modulator.
 122. The method of claim 121, wherein the AhR modulator is an AhR antagonist.
 123. A method of making a platelet, comprising culturing a MEP in the presence of an AhR modulator to make a Mk and culturing the Mk under conditions sufficient for differentiation of a platelet.
 124. The method of claim 123, wherein the AhR modulator is an AhR antagonist.
 125. The method of claim 123, further comprising culturing the MEP in megakaryocyte specification media.
 126. A method of making a platelet comprising culturing an Mk in the presence of an AhR modulator.
 127. The method of claim 126, wherein the AhR modulator is an AhR antagonist.
 128. The method of claim 127, wherein the AhR antagonist increases the rate of production of proplatelets in the culture.
 129. A method of making a transfusion composition, comprising providing platelets made by the method of claim 117 and combining the platelets with a composition comprising at least one of an anticoagulant, a buffer, and a nutrient, to thereby provide the transfusion composition.
 130. A method of making a transfusion composition, comprising providing platelets made by the method of claim 123 and combining the platelets with a composition comprising at least one of an anticoagulant, a buffer, and a nutrient, to thereby provide the transfusion composition.
 131. A method of making a transfusion composition, comprising providing platelets made by the method of claim 126 and combining the platelets with a composition comprising at least one of an anticoagulant, a buffer, and a nutrient, to thereby provide the transfusion composition.
 132. A method of providing platelets to a patient in need thereof, comprising providing a transfusion composition according to claim 129, and transfusing the transfusion composition into the circulatory system of the patient.
 133. A method of providing platelets to a patient in need thereof, comprising providing a transfusion composition according to claim 130, and transfusing the transfusion composition into the circulatory system of the patient.
 134. A method of providing platelets to a patient in need thereof, comprising providing a transfusion composition according to claim 131, and transfusing the transfusion composition into the circulatory system of the patient.
 135. The method of claim 132, wherein the platelets are differentiated from induced pluripotent stem cells derived from somatic cells of the patient.
 136. A method of screening a compound for an effect on a platelet, comprising: a) providing a platelet according to claim 117; b) contacting the platelet with the compound; and c) observing a change in the platelet.
 137. A method of screening a compound for an effect on a platelet, comprising: a) providing a platelet according to claim 123; b) contacting the platelet with the compound; and c) observing a change in the platelet.
 138. A method of screening a compound for an effect on a platelet, comprising: a) providing a platelet according to claim 126; b) contacting the platelet with the compound; and c) observing a change in the platelet. 